Human methionine synthase: cloning, and methods for evaluating risk of neural tube defects, cardiovascular disease, and cancer

ABSTRACT

The invention features a method for detecting an increased likelihood of hyperhomocysteinemia and, in turn, an increased or decreased likelihood of neural tube defects or cardiovascular disease. The invention also features therapeutic methods for reducing the risk of neural tube defects, colon cancers and related cancers. Also provided are the sequences of the human methionine synthase gene and protein and compounds and kits for performing the methods of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims priority from U.S. Provisional ApplicationsSerial Nos. 60/031,964 and 60/050,310, filed Nov. 27, 1996 and Jun. 20,1997, respectively.

FIELD OF THE INVENTION

[0002] The invention relates to the diagnosis and treatment of patientsat risk for methionine synthase deficiency and associated altered riskfor diseases such as neural tube defects, cardiovascular disease, andcancer.

BACKGROUND OF THE INVENTION

[0003] Methionine synthase (EC 2.1.1.13,5-methyltetrahydrofolate-homocysteine methyltransferase) catalyses theremethylation of homocysteine to methionine in a reaction in whichmethylcobalamin serves as an intermediate methyl carrier. This occurs bytransfer of the methyl group of 5-methyltetrahydrofolate to theenzyme-bound cob(I)alamin to form methylcobalamin with subsequenttransfer of the methyl group to homocysteine to form methionine. Overtime, cob(I)alamin may become oxidized to cob(II)alamin rendering theenzyme inactive. Regeneration of the functional enzyme occurs throughthe methionine synthase-mediated methylation of the cob(II)alamin inwhich S-adenosylmethionine is utilized as methyl donor. In E. coli, twoflavodoxins have been implicated in the reductive activation ofmethionine synthase (Fujii, K. and Huennekens, F. M. (1974) J. Biol.Chem., 249, 6745-6753). A methionine synthase-linked reducing system hasyet to be identified in mammalian cells.

[0004] Deficiency of methionine synthase activity results inhyperhomocysteinemia, homocystinuria, and megaloblastic anemia withoutmethylmalonic aciduria (Rosenblatt, D. S. (1995) The Metabolic andMolecular Bases of Inherited Disease. McGraw-Hill, New York, pp.3111-3128; Fenton, W. A. and Rosenberg, L. E. (1995) The Metabolic andMolecular Bases of Inherited Disease. McGraw-Hill, New York, pp.3129-3149). Two classes of methionine synthase-associated geneticdiseases have been proposed based on complementation experiments betweenpatient fibroblast cell lines (Watkins, D. and Rosenblatti D. S. (1988)J. Clin. Invest., 81, 1690-1694). One complementation group, cblE, hasbeen postulated to be due to deficiency of the reducing system requiredfor methionine synthesis (Rosenblatt, D. S., Cooper, B. A., Pottier, A.,Lue-Shing, H., Matiaszuk, N. and Grauer, K. (1984) J. Clin. Invest., 74,2149-2156). Cells from patients in the cblE group fail to incorporate¹⁴C-methyltetrahydrofolate into methionine in whole cells but havesignificant methionine synthase activity in cell extracts in thepresence of a potent reducing agent. The second complementation group,cblG group, is thought to result from defects of the methionine synthaseapoenzyme. Mutant cells from this group show deficient methioninesynthase activity in both whole cells and cell extracts (Watkins, D. andRosenblatt, D. S. (1988) J. Clin. Invest., 81, 1690-1694; Watkins, D.and Rosenblatt, D. S. (1989) Am. J Med. Genet., 34, 427-434). Moreover,some cblG patients show defective binding of cobalamin to methioninesynthase in cells incubated with radiolabelled cyanocobalamin (Sillaots,S. L., Hall, C. A., Hurteloup, V., and Rosenblatt, D. S. (1992) Biochem.Med. Metab. Biol., 47, 242-249).

[0005] The cobalamin-dependent methionine synthase of E. coli has beencrystallized and the structure of its active site determined(Luschinsky, C. L., Drummond, J. T., Matthews, R. G., and Ludwig, M. L.(1992) J. Molec. Biol., 225, 557-560; Drennan, C. L., Huang, S.,Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science, 266,1669-1674.). The gene encoding methionine synthase has not been clonedfrom mammals.

SUMMARY OF THE INVENTION

[0006] We have cloned a gene for mammalian methionine synthase fromhumans and discovered that mutations in this gene are associated withhyperhomocysteinemia. Hyperhomocysteinemia is a condition that has beenimplicated in cardiovascular disease and neural tube defects. Thepresence of such mutations in methionine synthase gene are, thus,associated with increased risk for cardiovascular disease, altered riskfor neural tube defects, and decreased risk of colon cancer. Theinvention features methods for risk detection and treatment of patientswith hyperhomocysteinemia, cardiovascular disease, neural tube defects,and cancer. The invention also features compounds and kits which may beused to practice the methods of the invention, methods and compounds fortreating or preventing these conditions and methods of identifyingtherapeutics for the treatment and prevention of these conditions.

[0007] In the first aspect, the invention provides purified wild-typemammalian methionine synthase gene, and mutated and polymorphic versionsof the mammalian methionine synthase gene, fragments of the wild-type,mutated, and polymorphic gene, and sense and antisense sequences whichmay be used in the methods of the invention. Preferably, the gene ishuman. The proteins encoded therefrom are also an aspect of theinvention as is a methionine synthase polypeptide having conservativesubstitutions. Preferably, the protein is a recombinant or purifiedprotein having a mutation conferring hypeihomocysteinemia when presentin a mammal. In addition, nucleic acids, including genomic DNA, mRNA,and cDNA, and the nucleic acid set forth in SEQ ID NO: 1, or degeneratevariants thereof, are provided. The shorter nucleic acid sequences areappropriate for use in cloning, characterizing mutations, theconstruction of mutations, and creating deletions. In one embodiment,the nucleic acid set forth in SEQ ID NO: 1 is a probe that hybridizes athigh stringency to sequences found within the nucleic acid of SEQ IDNO: 1. In further embodiments, the probe has a sequence complementary toat least 50% of at least 60 nucleotides, or the sequence iscomplementary to at least 90% of at least 18 nucleotides. Proteinfragments also are provided. The shorter peptides may be used, forexample, in the generation of antibodies to the methionine synthaseprotein. In some embodiments of this aspect of the invention nucleicacid fragments useful for detection of mutations in the region of themethionine synthase gene which encodes the cobalamin binding domain, andfor detecting those mutations which indicate an increased likelihood ofhyperhomocysteinemia, are preferred. Most preferred fragments are thoseuseful for detecting the 2756 A→G, Δbp 2640-2642, and 2758 C→Gmutations/polymorphisms. Given Applicants' discovery, one skilled in theart may readily determine which nucleic acids, detection methods, andmutations are most useful. Mutant proteins encoded by these mutations,including, but not limited to, H920D, ΔIle 881, and D919G are alsoprovided by the invention. Such mutant and polymorphic polypeptides mayhave decreased or increased biological activity, relative to wild-typemethionine synthase.

[0008] In a related aspect, the invention provides antibodies thatspecifically bind mammalian methionine synthase, and a method forgenerating such an antibody. The antibody may specifically bind awild-type methionine synthase, or a mutant or polymorphic methioninesynthase. A method for detecting a wild-type, mutant, or polymorphicmethionine synthase using the antibody is also provided by theinvention.

[0009] In a second aspect, the invention provides a method for detectingan increased or decreased risk for hyperhomocysteinemia in a fetus orindividual patient. Such a fetus or patient is at increased or decreasedrisk for neural tube defects and/or cardiovascular disease and at adecreased risk of developing colon cancer. The method includes detectionof mutations in the methionine synthase gene present in the fetus, theindividual patient, and/or the blood relatives of the fetus and patient.The presence of mutations, particularly in the cobalamin binding domain,indicate an altered (e.g., increased or decreased) risk ofhyperhomocysteinemia, neural tube defects, cancer, and cardiovasculardisease.

[0010] In a related aspect, the invention provides kits for thedetection of mutations in the human methionine synthase gene. Such kitsmay include, for example, nucleic acid sequences, including probes,useful for PCR, SSCP, or RFLP detection of such mutations. Antibodiesspecific for proteins having mutations, correlated with an increasedlikelihood of hyperhomocysteinemia, may also be included in the kits ofthe invention.

[0011] In a fourth aspect, the invention features a method for screeningfor compounds which alter methionine synthase expression or ameliorateor exacerbate conditions of hyperhomocysteinemia. In variousembodiments, the invention includes monitoring mutant or wild-typemammalian methionine synthase biological activity by monitoringmethionine synthase enzymatic activity, or monitoring methioninesynthase gene expression levels, by monitoring methionine synthase genetranscription, RNA stability, RNA translation and/or protein stability.In preferred embodiments the methionine synthase gene or protein beingmonitored is a gene or protein having a mutation associated withhyperhomocysteinemia, and samples are selected from purifed or partiallypurified methionine synthase, cell lysate, a cell, or an animal.Standard assay techniques known to those skilled in the art may beemployed in the various embodiments. Compounds detected using thisscreen can be used to prevent or treat cardiovascular disease and neuraltube defects or, in the alternative, to prevent or treat colon cancer.Kits for performing the above screens are also a part of the invention.

[0012] In a related aspect, the invention provides nucleic acidsencoding wild-type, polymorphic, and mutated methionine synthase, inwhich the nucleic acid is operably linked to regulatory sequences,comprising a promoter, for the expression of the encoded polypeptides.In one embodiment, the promoter is inducible. The invention alsoprovides cells, including prokaryotic and eukaryotic cells, comprisingthe nucleic acids. The eukaryotic cells may be yeast cells or mammaliancells.

[0013] In another related aspect, the invention features a transgenicmammal having a methionine synthase transgene. The gene may bewild-type, or may contain a mutation or polymorphism. The mammal mayhave a mutation associated with hyperhomocysteinemia in its methioninesynthase gene in an expressible genetic construction or may have adeletion or knockout mutation in one or both alleles sufficient toabolish methionine synthase expression from the locus. In addition, oras a replacement, the mammal may have the methionine synthase gene fromanother species. For example, in one preferred embodiment the transgenicmammal is a rodent such as a mouse and the transgene is from a human.Cells from these transgenic or knockout animals are also provided by theinvention. Such transgenic mammals may be used to screen for drugs forthe treatment of diseases related to hyperhomocysteinemia.

[0014] In a sixth aspect, the invention features a method for treatingpatients with neural tube defects, colon cancer or related cancers bythe delivery of antisense methionine synthase nucleic acid sufficient tolower the levels of methionine synthase polypeptide biological activity.

[0015] In a related aspect, the invention provides a method for treatingor preventing cardiovascular disease, neural tube defects and cancer.The method comprises detecting an altered risk of such defects byanalyzing methionine synthase nucleic acid, potential test subjectsbeing a mammal, a potential parent, either male or female, a pregnantmammal, or a developing embryo or fetus, and then by exposing thesubject (e.g., patient or pregnant mammal) to metabolites or cofactorssuch as, but not limited to, folate, cobalamin, S-adenosyl methionine,betaine, or methionine. In another related aspect, the inventionfeatures a method of pretreating or treating colon cancer or neural tubedefects by inhibiting or activating methionine synthase biologicalactivity in a mammal, pregnant mammal, embryo, or fetus. In preferredembodiments, this inhibiting or activating may be effected by exposingthe subject to nucleic acids, peptides or small molecule-basedinhibitors or activators of methionine synthase or substrates. Theexposure is to quantities of the compound sufficient to reduce theprobability of the subject developing the disease or to confer anincreased likelihood of a decrease in the disease symptoms of thesubject.

[0016] By “methionine synthase,” “methionine synthase protein,” or“methionine synthase polypeptide” is meant a polypeptide, or fragmentthereof, which has at least 50% amino acid identity to boxes 1-4 of thehuman methionine synthase polypeptide (SEQ ID NO: 2) (see FIG. 1). It isunderstood that polypeptide products from splice variants of methioninesynthase gene sequences are also included in this definition.Preferably, the methionine synthase protein is encoded by nucleic acidhaving a sequence which hybridizes to a nucleic acid sequence present inSEQ ID NO: 1 (human methionine synthase cDNA) under stringentconditions. Even more preferably the encoded polypeptide also hasmethionine synthase biological activity.

[0017] By “methionine synthase nucleic acid” or “methionine synthasegene” is meant a nucleic acid, such as genomic DNA, cDNA, or mRNA, thatencodes methionine synthase, a methionine synthase protein, methioninesynthase polypeptide, or portion thereof, as defined above. A methioninesynthase nucleic acid also may be a methionine synthase primer or probe,or antisense nucleic acid that is complementary to a methionine synthasenucleic acid.

[0018] By “wild-type methionine synthase” is meant a methionine synthasenucleic acid or methionine synthase polypeptide having the nucleic acidand/or amino acid sequence most often observed among members of a givenanimal species and not statistically associated with a diseasephenotype. Wild-type methionine synthase is biologically activemethionine synthase. A wild-type methionine synthase is, for example, ahuman methionine synthase polypeptide having the sequence of SEQ ID NO:1.

[0019] By “mutant methionine synthase,” “methionine synthasemutation(s),” “mutations in methionine synthase,” “polymorphicmethionine synthase,” “methionine synthase polymorphism(s),”“polymorphisms in methionine synthase,” is meant a methionine synthasepolypeptide or nucleic acid having a sequence that deviates from thewild-type sequence in a manner sufficient to confer an altered risk fora disease phenotype, or enhanced protection against a disease, in atleast some genetic and/or environmental backgrounds. Such is mutationsmay be naturally occurring or artificially induced. They may be, withoutlimitation, insertion, deletion, frameshift, or missense mutations. Amutant methionine synthase protein may have one or more mutations, andsuch mutations may affect different aspects of methionine synthasebiological activity (protein function), to various degrees.Alternatively, a methionine synthase mutation may indirectly affectmethionine synthase biological activity by influencing, for example, thetranscriptional activity of a gene encoding methionine synthase, or thestability of methionine synthase mRNA. For example, a mutant methioninesynthase gene may be a gene which expresses a mutant methionine synthaseprotein or may be a gene which alters the level of methionine synthaseprotein in a manner sufficient to confer a disease phenotype in at leastsome genetic and/or environmental backgrounds.

[0020] By “biologically active” methionine synthase is meant amethionine synthase protein or methionine synthase gene that provides atleast one biological function equivalent to that of the wild-typemethionine synthase polypeptide or methionine synthase gene. Biologicalactivities of a methionine synthase polypeptide include, and are notlimited to, the ability to catalyze the methylation of homocysteine togenerate methionine. Preferably, a biologically active methioninesynthase will display activity equivalent to at least 35% of wild-typeactivity, more preferably, a biologically active methionine synthasewill display at least 40-55% of wild-type activity, still morepreferably, a biologically active methionine synthase will display atleast 60-75% of wild-type activity, and most preferably, a biologicallyactive methionine synthase will display at least 80-90% of wild-typeactivity. A biologically active methionine synthase also may displaymore than 100% of wild-type activity. Preferably, the biologicalactivity of the wild-type methionine synthase is determined using themethionine synthase nucleic acid of SEQ ID NO: 1 or methionine synthasepolypeptide of SEQ ID NO: 2. The degree of methionine synthasebiological activity may be intrinsic to the methionine synthasepolypeptide itself, or may be modulated by increasing or decreasing thenumber of methionine synthase polypeptide molecules presentintracellularly.

[0021] By “high stringency conditions” is meant hybridization in 2×SSCat 40° C. with a DNA probe length of at least 40 nucleotides. For otherdefinitions of high stringency conditions, see F. Ausubel et al.,Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley &Sons, New York, N.Y., 1994, hereby incorporated by reference.

[0022] By “analyzing” or “analysis” is meant subjecting a methioninesynthase nucleic acid or methionine synthase polypeptide to a testprocedure that allows the determination of whether a methionine synthasegene is wild-type or mutant. For example, one could analyze themethionine synthase genes of an animal by amplifying genomic DNA usingthe polymerase chain reaction, and then determining the DNA sequence ofthe amplified DNA.

[0023] By “probe” or “primer” is meant a single- or double-stranded DNAor RNA molecule of defined sequence that can base pair to a second DNAor RNA molecule that contains a complementary sequence (the “target”).The stability of the resulting hybrid depends upon the extent of thebase pairing that occurs. The extent of base-pairing is affected byparameters such as the degree of complementarity between the probe andtarget molecules, and the degree of stringency of the hybridizationconditions. The degree of hybridization stringency is affected byparameters such as temperature, salt concentration, and theconcentration of organic molecules such as formamide, and is determinedby methods known to one skilled in the art Probes or primers specificfor methionine synthase nucleic acid preferably will have at least 35%sequence identity, more preferably at least 45-55% sequence identity,still more preferably at least 60-75% sequence identity, still morepreferably at least 80-90% sequence identity, and most preferably 100%sequence identity. Probes may be detectably-labelled, eitherradioactively, or non-radioactively, by methods well-known to thoseskilled in the art. Probes are used for methods involving nucleic acidhybridization, such as: nucleic acid sequencing, nucleic acidamplification by the polymerase chain reaction, single strandedconformational polymorphism (SSCP) analysis, restriction fragmentpolymorphism (RFLP) analysis, Southern hybridization, Northernhybridization, in situ hybridization, electrophoretic mobility shiftassay (EMSA).

[0024] By “pharmaceutically acceptable carrier” means a carrier which isphysiologically acceptable to the treated mammal while retaining thetherapeutic properties of the compound with which it is administered.One exemplary pharmaceutically acceptable carrier is physiologicalsaline. Other physiologically acceptable carriers and their formulationsare known to one skilled in the art and described, for example, inRemington's Pharmaceutical Sciences, (18^(th) edition), ed. A. Gennaro,1990,, Mack Publishing Company, Easton, Pa.

[0025] By “substantially identical” is meant a polypeptide or nucleicacid exhibiting at least 50%, preferably 85%, more preferably 90%, andmost preferably 95% identity to a reference amino acid or nucleic acidsequence. For polypeptides, the length of comparison sequences willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids. For nucleic acids, the length of comparison sequences willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides.

[0026] Sequence identity is typically measured using sequence analysissoftware with the default parameters specified therein (e.g., SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705). This software program matches similar sequences by assigningdegrees of homology to various substitutions, deletions, and othermodifications. Conservative nucleotide substitutions typically includesubstitutions which generate changes within the following groups:glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamicacid, asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine.

[0027] By “substantially pure polypeptide” is meant a polypeptide thathas been separated from the components that naturally accompany it.Typically, the polypeptide is substantially pure when it is at least60%, by weight, free from the proteins and naturally-occurring organicmolecules with which it is naturally associated. Preferably, thepolypeptide is a methionine synthase polypeptide that is at least 75%,more preferably at least 90%, and most preferably at least 99%, byweight, pure. A substantially pure methionine synthase polypeptide maybe obtained, for example, by extraction from a natural source (e.g., afibroblast or liver cell) by expression of a recombinant nucleic acidencoding a methionine synthase polypeptide, or by chemicallysynthesizing the protein. Purity can be measured by any appropriatemethod, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

[0028] A protein is substantially free of naturally associatedcomponents when it is separated from those contaminants which accompanyit in its natural state. Thus, a protein which is chemically synthesizedor produced in a cellular system different from the cell from which itnaturally originates will be substantially free from its naturallyassociated components. Accordingly, substantially pure polypeptides notonly includes those derived from eukaryotic organisms but also thosesynthesized in E. coli or other prokaryotes.

[0029] By “substantially pure DNA” is meant DNA that is free of thegenes which, in the naturally-occurring genome of the organism fromwhich the DNA of the invention is derived, flank the gene. The termtherefore includes, for example, a recombinant DNA which is incorporatedinto a vector; into an autonomously replicating plasmid or virus; orinto the genomic DNA of a prokaryote or eukaryote; or which exists as aseparate molecule (e.g., a cDNA or a genomic or cDNA fragment producedby PCR or restriction endonuclease digestion) independent of othersequences. It also includes a recombinant DNA which is part of a hybridgene encoding additional polypeptide sequence.

[0030] By “transgene” is meant any piece of DNA which is inserted byartifice into a cell, and becomes part of the genome of the organismwhich develops from that cell. Such a transgene may include a gene whichis partly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism.

[0031] By “transgenic” is meant any cell which includes a DNA sequencewhich is inserted by artifice into a cell and becomes part of the genomeof the organism which develops from that cell. As used herein, thetransgenic organisms are generally transgenic mammals (e.g., rodentssuch as rats or mice) and the DNA (transgene) is inserted by artificeinto the nuclear genome. Preferably the inserted DNA encodes a proteinin at least some cells of the organism.

[0032] By “knockout mutation” is meant an alteration in the nucleic acidsequence that reduces the biological activity of the polypeptidenormally encoded therefrom by at least 80% relative to the unmutatedgene. The mutation may, without limitation, be an insertion, deletion,frameshift mutation, or a missense mutation. Preferably, the mutation isan insertion or deletion, or is a frameshift mutation that creates astop codon.

[0033] By “transformation” is meant any method for introducing foreignmolecules into a cell. Lipofection, DEAE-dextran-mediated transfection,microinjection, protoplast fusion, calcium phosphate precipitation,retroviral delivery, electroporation, and biolistic transformation arejust a few of the methods known to those skilled in the art which may beused. For example, biolistic transformation is a method for introducingforeign molecules into a cell using velocity driven microprojectilessuch as tungsten or gold particles. Such velocity-driven methodsoriginate from pressure bursts which include, but are not limited to,helium-driven, air-driven, and gunpowder-driven techniques. Biolistictransformation may be applied to the transformation or transfection of awide variety of cell types and intact tissues including, withoutlimitation, intracellular organelles (e.g., and mitochondria andchloroplasts), bacteria, yeast, fungi, algae, animal tissue, andcultured cells.

[0034] By “transformed cell” is meant a cell into which (or into anancestor of which) has been introduced, by means of recombinant DNAtechniques, a DNA molecule encoding (as used herein) a methioninesynthase polypeptide.

[0035] By “positioned for expression” is meant that the DNA molecule ispositioned adjacent to a DNA sequence which directs transcription andtranslation of the sequence (i.e., facilitates the production of, e.g.,a methionine synthase polypeptide, a recombinant protein or a RNAmolecule).

[0036] By “promoter” is meant a minimal sequence sufficient to directtranscription. Also included in the invention are those promoterelements which are sufficient to render promoter-dependent geneexpression controllable for cell type-specific, tissue-specific,temporal-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ or intron sequence regions ofthe native gene.

[0037] By “operably linked” is meant that a gene and one or moreregulatory sequences are connected in such a way as to permit geneexpression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequences.

[0038] By “conserved region” is meant any stretch of six or morecontiguous amino acids exhibiting at least 30%, preferably 50%, and mostpreferably 70% amino acid sequence identity between two or more of themethionine synthase family members, (e.g., between human and bacterialmethionine synthase). Examples of conserved regions within methioninesynthase are Boxes 1-4 (FIG. 1).

[0039] By “detectably-labeled” is meant any means for marking andidentifying the presence of a molecule, e.g., an oligonucleotide probeor primer, a gene or fragment thereof, or a cDNA molecule. Methods fordetectably-labeling a molecule are well known in the art and include,without limitation, radioactive labeling (e.g., with an isotope such as³²P or ³⁵S) and nonradioactive labeling (e.g., chemiluminescentlabeling, e.g., fluorescein labeling).

[0040] By “antisense” as used herein in reference to nucleic acids, ismeant a nucleic acid sequence that is complementary to the coding strandof a gene, preferably, a methionine synthase gene. An antisense nucleicacid is capable of preferentially lowering the activity of a mutantmethionine synthase polypeptide encoded by a mutant methionine synthasegene.

[0041] By “purified antibody” is meant antibody which is at least 60%,by weight, free from proteins and naturally occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably 90%, and most preferably at least 99%, byweight, antibody, e.g., a methionine synthase amino-terminus-specificantibody. A purified antibody may be obtained, for example, by affinitychromatography using recombinantly-produced protein or conserved motifpeptides and standard techniques.

[0042] By “specifically binds” is meant an antibody that recognizes andbinds a human methionine synthase polypeptide but that does notsubstantially recognize and bind other non-methionine synthase moleculesin a sample, e.g., a biological sample, that naturally includes protein.A preferred antibody binds to the methionine synthase polypeptidesequence of SEQ ID NO: 2 (FIG. 3).

[0043] By “neutralizing antibodies” is meant antibodies that interferewith any of the biological activities of a wild-type or mutantmethionine synthase polypeptide, for example, the ability of methioninesynthase to catalyze the transfer of a methyl group to homocysteine. Theneutralizing antibody may reduce the ability of a methionine synthasepolypeptide to catalyze the transfer preferably by 10% or more, morepreferably by 25% or more, still more preferably by 50% or more, yetpreferably by 70% or more, and most preferably by 90% or more.

[0044] Any standard assay for the biological activity of methioninesynthase, may be used to assess potentially neutralizing antibodies thatare specific for methionine synthase.

[0045] By “expose” is meant to allow contact between an animal, cell,lysate or extract derived from a cell, or molecule derived from a cell,and a test compound.

[0046] By “treat” is meant to submit or subject an animal (e.g. ahuman), cell, lysate or extract derived from a cell, or molecule derivedfrom a cell to a test compound.

[0047] By “test compound” is meant a chemical, be it naturally-occurringor artificially-derived, that is surveyed for its ability to modulate analteration in reporter gene activity or protein levels, by employing oneof the assay methods described herein. Test compounds may include, forexample, peptides, polypeptides, synthesized organic molecules,naturally occurring organic molecules, nucleic acid molecules, andcomponents thereof.

[0048] By “assaying” is meant analyzing the effect of a treatment, be itchemical or physical, administered to whole animals or cells derivedtherefrom. The material being analyzed may be an animal, a cell, alysate or extract derived from a cell, or a molecule derived from acell. The analysis may be for the purpose of detecting altered proteinbiological activity, altered protein stability, altered protein levels,altered gene expression, or altered RNA stability. The means foranalyzing may include, for example, for example, the detection of theproduct of an enzymatic reaction, (e.g., the formation of methionine asa result of methionine synthase activity), antibody labeling,immunoprecipitation, and methods known to those skilled in the art fordetecting nucleic acids.

[0049] By “modulating” is meant changing, either by decrease orincrease, in biological activity.

[0050] By “a decrease” is meant a lowering in the level of biologicalactivity, as measured by a lowering/increasing of: a) the formation ofmethionine as a result of methionine synthase activity; b) protein, asmeasured by ELISA; c) reporter gene activity, as measured by reportergene assay, for example, lacZ/β-galactosidase, green fluorescentprotein, luciferase, etc.; or d) mRNA, levels of at least 30%, asmeasured by PCR relative to an internal control, for example, a“housekeeping” gene product such as β-actin or glyceraldehyde3-phosphate dehydrogenase (GAPDH) or an externally added nucleic acidstandard. In all cases, the lowering is preferably by at least 10% morepreferably by at least 25%, still more preferably by at least 50%, andeven more preferably by at least 70%.

[0051] By “an increase” is meant a rise in the level of biologicalactivity, as measured by a lowering/increasing of: a) the formation ofmethionine as a result of methionine synthase activity; b) protein, asmeasured by ELISA; c) reporter gene activity, as measured by reportergene assay, for example, lacZ/β-galactosidase, green fluorescentprotein, luciferase, etc.; or d) mRNA, levels of at least 30%, asmeasured by PCR relative to an internal control, for example, a“housekeeping” gene product such as β-actin or glyceraldehyde3-phosphate dehydrogenase (GAPDH) or an externally added nucleic acidstandard. Preferably, the increase is by 10% or more, more preferably by25% or more, even more preferably by 2-fold, and most preferably by atleast 3-fold.

[0052] By “alteration in the level of gene expression” is meant a changein gene activity such that the amount of a product of the gene, i.e.,mRNA or polypeptide, is increased or decreased, or that the stability ofthe mRNA or the polypeptide is increased or decreased.

[0053] By “reporter gene” is meant any gene which encodes a productwhose expression is detectable and/or quantitatable by immunological,chemical, biochemical or biological assays. A reporter gene product may,for example, have one of the following attributes, without restriction:fluorescence (e.g., green fluorescent protein), enzymatic activity(e.g., lacZ/β-galactosidase, luciferase, chloramphenicolacetyltransferase), toxicity (e.g., ricin A), or an ability to bespecifically bound by a second molecule (e.g., biotin or a detectablylabelled antibody). It is understood that any engineered variants ofreporter genes, which are readily available to one skilled in the art,are also included, without restriction, in the forgoing definition.

[0054] By “protein” or “polypeptide” or “polypeptide fragment” is meantany chain of more than two amino acids, regardless of post-translationalmodification (e.g., glycosylation or phosphorylation), constituting allor part of a naturally-occurring polypeptide or peptide, or constitutinga non-naturally occurring polypeptide or peptide.

[0055] By “missense mutation” is meant the substitution of one purine orpyrimidine base (i.e. A, T, G, or C) by another within a nucleic acidsequence, such that the resulting new codon encodes an amino aciddistinct from the amino acid originally encoded by the reference (e.g.wild-type) codon.

[0056] By “deletion mutation” is meant the deletion of at least onenucleotide within a polynucleotide coding sequence. A deletion mutationalters the reading frame of a coding region unless the deletion consistsof one or more contiguous 3-nucleotide stretches (i.e. “codons”).Deletion of a codon from a nucleotide coding region results in thedeletion of an amino acid from the resulting polypeptide.

[0057] By “frameshift mutation” is meant the insertion or deletion of atleast one nucleotide within a polynucleotide coding sequence. Aframeshift mutation alters the codon reading frame at and/or downstreamfrom the mutation site. Such a mutation results either in thesubstitution of the encoded wild-type amino acid sequence by a novelamino acid sequence, or a premature termination of the encodedpolypeptide due to the creation of a stop codon, or both.

DETAILED DESCRIPTION OF THE DRAWINGS

[0058] The drawings will first be briefly described.

[0059]FIG. 1 is a diagram showing four homologous regions amongmethionine synthases. Boxes 1 to 4 were used to design degenerateoligonucleotides for the initial cloning experiments. Ec: Escherichiacoli, accession number J04975; Ss: Synechocystis sp., accession numberD64002; Ml1 and Ml2: Mycobacterium leprae, accession number U000175 (seeDrennan et al., 1994); Hi: Haemophilus influenzae, accession numberU32730; Ce: Caenorhabditis elegans, accession number Z46828; Hs: Homosapiens, this work. Identical residues are indicated by a star above thealignment. Amino acid position for each protein is shown at left.

[0060]FIG. 2 is a diagram showing overlapping PCR fragments generated toclone human methionine synthase. Oligonucleotides are described in Table1.

[0061] Primers in parentheses designate mispriming outcomes thatgenerated valid internal sequence. iPCRc: inverse PCR on cDNA, iPCRg:inverse PCR on genomic DNA.

[0062]FIG. 3 is a diagram showing nucleotide sequence (SEQ ID NO: 1) anddeduced amino acid sequence (SEQ ID NO: 2) of human methionine synthase.The nucleotide residue numbering is shown in the left margin, and theamino acid residue numbering is shown in the right margin.

[0063]FIG. 4 is a photograph showing mapping of the human methioninesynthase gene using FISH. Signals are clearly visible at 1q43 (arrows).

[0064] FIGS. 5A-5C is a series of photographs showing diagnostic testsfor mutations in the methionine synthase gene. Numbers above the gellanes correspond to patients cell lines whereas the letter “c”identifies wild-type controls. FIG. 5A: HaeIII restriction analysis ofgenomic DNA PCR products using primers #1796 and #305A. The 2756A-Gchange creates a HaeIII site. Expected fragments, 2756A allele: 189 bp,2756G allele: 159 and 30 bp (the 30 bp fragment was run off the gel).FIG. 5B: Heteroduplex analysis of PCR products amplified from RTreactions of patient 1892 and 3 controls. RT-PCR was done with primers#1772 and #1773. Expected PCR product: 338 bp, heteroduplexes can beseen above this band in patient 1892 (heterozygous for Δ2640-2642). C.Sau96I restriction analysis of genomic DNA PCR products. PCR was done asin (A). The 2758C→G mutation abolishes a Sau96I restriction endonucleasesite in patient 2290. Expected fragments, control allele: 159, 30 bp,mutant allele: 189 bp (the 30 bp fragment has been run off the gel).

[0065]FIG. 6. shows an amino acid sequence comparison among methioninesynthases in the Box 2 region. Identical residues are indicated by astar above the alignment. Dots show partially conserved residues, forwhich at least {fraction (6/7)} identical or similar residues can bealigned (A,G,S,T; D,E,N,Q; V,L,I,M; K,R; and F,W,Y (Bordo,D. andArgos,P. (1991) J. Molec. Biol., 217, 721-729)). Mutations identified inthis work are shown below the alignment. For abbreviations, see FIG. 1;Mm: Mus musculus. The seven amino acids conserved in cobalamin-bindingproteins (Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G.,and Ludwig, M. L. (1994) Science, 266, 1669-1674) are underlined.

DETAILED DESCRIPTION

[0066] We used specific regions of homology within the methioninesynthase sequences of several lower organisms to clone a humanmethionine synthase cDNA (SEQ ID NO:1) by a combination of RT-PCR andinverse PCR. The enzyme (SEQ ID NO:2) is 1265 amino acids in length andcontains the seven residue structure-based sequence fingerprintidentified for cobalamin-containing enzymes. The gene was localized tochromosome 1q43 by the FISH technique. We have identified one missensemutation and a 3 base pair deletion in patients of the cblGcomplementation group of inherited homocysteine/folate disorders by SSCPand sequence analysis, as well as an amino acid substitution present inhigh frequency in the general population.

[0067] We conclude that the cDNA that we have identified corresponds tohuman methionine synthase on the basis of homology to known methioninesynthases and by the identification of mutations in patients with adeficiency of enzyme activity. The most striking sequence conservationwas found in four boxes of 9-13 amino acids. Box 2 has been proposed tocorrespond to part of the cobalamin binding domain (Drennan, C. L.,Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994)Science, 266, 1669-1674). It contains 13 consecutive residues that areidentical in all known methionine synthases. Three amino acids withinbox 2 and four others C-terminal to it correspond to residues proposedby Drennan et al. (Drennan, C. L., Huang, S., Drummond, J. T., Matthews,R. G., and Ludwig, M. L. (1994) Science, 266, 1669-1674) as astructure-based sequence fingerprint for cobalamin binding. The threeamino acids appear to make direct contact with the lower face of thecorrin ring and dimethylbenzimidazole tail of cobalamin, determined fromthe crystal structure of the E. coli enzyme at 3 Å resolution (Drennan,C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L.(1994) Science, 266, 1669-1674). All seven residues are identical in thehuman sequence (FIG. 6).

[0068] A survey of the NCBI databases for homology to the humanmethionine synthase using BLASTP yielded the various methioninesynthases listed in FIG. 1, as well as the glutamate mutase (S41332,Q05488) and methylmalonyl-CoA mutase (P11653)(adenosyl-cobalamindependent mutases) used to deduce the sequence fingerprint for cobalaminbinding (Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G.,and Ludwig, M. L. (1994) Science, 266, 1669-1674). Homology was alsofound with the cobalamin binding region of the corrinoid: coenzyme Mmethyltransferase of Methanosarcina barkeri (U36337), the5-methyltetrahydrofolate corrinoid/iron sulfur protein methyltransferaseof Clostridium thermoaceticum (L34780) and the B12-dependent2-methyleneglutarate mutase of Clostridium barkeri (S43552, S43237).Further, homology was found with the B12-binding site domain of therecently identified putative methionine synthase of Agrobacteriumtumefaciens (U48718; partial N-terminal sequence is given, up to regionof box 4). Significantly, homology with the B12-binding site domain wasalso found in the Hg resistance protein of Myxococcus xanthus (Z21955).This protein has not been described as having a cobalamin prostheticgroup.

[0069] The two mutations we have identified as candidates for causingcblG disease are located in the vicinity of the cobalamin binding domainby comparison with E. coli methionine synthase (FIG. 6). Ile881corresponds by sequence alignment to Val855 in the E. coli enzyme.Val855 is within a beta sheet strand that is part of an α/β domain thatis a variant of the Rossmann nucleotide binding fold. The H920Dsubstitution is found in a region which, in the E. coli enzyme, is in anα helix at the C-terminal end of the α/β domain. It is interesting thatthe polymorphism we have identified is at the adjacent residue (D919G).The functional role of the polymorphism and deleterious mutations willhave to be examined in expression experiments to confirm their preciseeffect on the protein.

[0070] Through the cloning of a cDNA for human methionine synthase andmutations therein, we can now determine the properties of the humanenzyme and complete the characterization of mutations in patients withsevere synthase deficiency. This analysis has allowed us to tiemutations in the gene to disturbances in homocysteine metabolism whichare known to result in hyperhomocysteinemia is a risk factor forcardiovascular disease (Boushey, C. J., Beresford, S. A., Omenn, G. S.,and Motulsky, A. G. (1995) JAMA, 274, 1049-1057) and neural tube defects(Steegers-Theunissen, R. P., Boers, G. H., Trijbels, F. J., Finkelstein,J. D., Blom, H. J., Thomas, C. M., Borm, G. F., Wouters, M. G., andEskes, T. K. (1994) Metab. Clin. Exp., 43, 1475-1480; and Mills, J. L.,McPartlin, J. M., Kirke, P. N., Lee, Y. J., Conley, M. R., Weir, D. G.and Scott, J. M. (1995) Lancet, 345, 149-151).

[0071] Our observations indicate the importance of methionine synthaseas one of several genes involved in homocysteine metabolism. Resultswith other pathway genes underscores the significance of our findings.For example, a recently-identified mutation in methylenetetrahydrofolatereductase, the enzyme that synthesizes the 5-methyltetrahydrofolatesubstrate for the methionine synthase reaction, results in mildhyperhomocysteinemia (Frosst,P., Blom,H. J., Milos,R., Goyette,P.,Sheppard,C. A., Matthews,R. G., Boers,G. J., den Heijer,M.,Kluijtmans,L. A., van den Heuvel,L. P., et al. (1995) Nat. Genet., 10,111-113). Evidence is accumulating that this mutation, present in 35-40%of alleles, is a risk factor in both cardiovascular disease and neuraltube defects (Rozen,R. (1996) Clin. Invest. Med., 19, 171-178). Webelieve that genetic variants of methionine synthase similarly lead tomild hyperhomocysteinemia with consequent impact on these twomultifactorial disorders.

[0072] We used specific regions of homology within the methioninesynthase sequences, including a portion of the cobalamin binding sitedetermined from the E. coli enzyme, to design degenerateoligonucleotides for RT-PCR-dependent cloning of human methioninesynthase. We confirmed the identification of the cDNA sequences forhuman methionine synthase by the high degree of homology to the enzymesin other species and the identification of mutations in patients fromthe cblG complementation group. We also mapped the gene to humanchromosome 1.

[0073] The assays described herein can be used to test for compoundsthat modulate methione synthase activity and hence may have therapeuticvalue in the prevention of neural tube defects, prevention and/ortreatment of colon cancer, cardiovascular disease, hyperhomocysteinemia,and megaloblastic anemia without methylmalonic aciduria.

Screens for Compounds that Modulate Methionine Synthase EnzymaticActivity

[0074] Screens for potentially useful therapeutic compounds thatmodulate methionine synthase activity may be readily performed, forexample, by assays that measure the incorporation of[14C]5-methyltetrahydrofolate into methionine or protein, or assays thatmeasure the conversion of [14C]-homocysteine into methionine or protein.Examples of such assays, which employ whole cells or cell lysates, arewell-known to those skilled in the art (see, e.g., Schuh, S., et al., N.Engl. J. Med. 1984, 310:686-69; Rosenblatt, D. S., et al., J. Clin.Invest. 1984, 74:2149-2156; Watkins, D., and Rosenblatt, D. S., J. Clin.Invest. 1988, 81:1690-1694; and Watkins, D., and Rosenblatt, D. S., Am.J. Med. Genet. 1989, 34:427-434), and may be readily adapted for highthroughput screening.

ELISA for the Detection of Compounds that Modulate Methionine SynthaseExpression

[0075] Enzyme-linked immunosorbant assays (ELISAs) are easilyincorporated into high-throughput screens designed to test large numbersof compounds for their ability to modulate levels of a given protein.When used in the methods of the invention, changes in a given proteinlevel of a sample, relative to a control, reflect changes in themethionine synthase expression status of the cells within the sample.Protocols for ELISA may be found, for example, in Ausubel et al.,CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1997.Lysates from cells treated with potential modulators of methioninesynthase expression are prepared (see, for example, Ausubel et al.,supra), and are loaded onto the wells of microtiter plates coated with“capture” antibodies specific for methionine synthase. Unbound antigenis washed out, and a methionine synthase-specific antibody, coupled toan agent to allow for detection, is added. Agents allowing detectioninclude alkaline phosphatase (which can be detected following additionof colorimetric substrates such as p-nitrophenolphosphate), horseradishperoxidase (which can be detected by chemiluminescent substrates such asECL, commercially available from Amersham) or fluorescent compounds,such as FITC (which can be detected by fluorescence polarization ortime-resolved fluorescence). The amount of antibody binding, and hencethe level of a methionine synthase polypeptide within a lysate sample,is easily quantitated on a microtiter plate reader.

[0076] As a baseline control for methionine synthase expression, asample that is not exposed to test compound is included. Housekeepingproteins are used as internal standards for absolute protein levels. Apositive assay result, for example, identification of a compound thatdecreases methionine synthase expression, is indicated by a decrease inmethionine synthase polypeptide within a sample, relative to themethionine synthase level observed in cells which are not treated with atest compound.

Reporter Gene Assays for Compounds that Modulate Methionine SynthaseExpression

[0077] Assays employing the detection of reporter gene products areextremely sensitive and readily amenable to automation, hence makingthem ideal for the design of high-throughput screens. Assays forreporter genes may employ, for example, colorimetric, chemiluminescent,or fluorometric detection of reporter gene products. Many varieties ofplasmid and viral vectors containing reporter gene cassettes are easilyobtained. Such vectors contain cassettes encoding reporter genes such aslacZ/β-galactosidase, green fluorescent protein, and luciferase, amongothers. Cloned DNA fragments encoding transcriptional control regions ofinterest (e.g. that of the mammalian methionine synthase gene) areeasily inserted, by DNA subcloning, into such reporter vectors, therebyplacing a vector-encoded reporter gene under the transcriptional controlof any gene promoter of interest. The transcriptional activity of apromoter operatively linked to a reporter gene can then be directlyobserved and quantitated as a function of reporter gene activity in areporter gene assay.

[0078] Cells are transiently- or stably-transfected with methioninesynthase control region/reporter gene constructs by methods that arewell known to those skilled in the art. Transgenic mice containingmethionine synthase control region/reporter gene constructs are used forlate-stage screens in vivo. Cells containing methioninesynthase/reporter gene constructs are exposed to compounds to be testedfor their potential ability to modulate methionine synthase expression.At appropriate timepoints, cells are lysed and subjected to theappropriate reporter assays, for example, a colorimetric orchemiluminescent enzymatic assay for lacZ/β-galactosidase activity, orfluorescent detection of GFP. Changes in reporter gene activity ofsamples treated with test compounds, relative to reporter gene activityof appropriate control samples, indicate the presence of a compound thatmodulates methionine synthase expression.

Quantitative PCR of Methionine Synthase mRNA as an Assay for Compoundsthat Modulate Methionine Synthase Expression

[0079] The polymerase chain reaction (PCR), when coupled to a precedingreverse transcription step (rtPCR), is a commonly used method fordetecting vanishingly small quantities of a target mRNA. When performedwithin the linear range, with an appropriate internal control target(employing, for example, a housekeeping gene such as actin), suchquantitative PCR provides an extremely precise and sensitive means ofdetecting slight modulations in mRNA levels. Moreover, this assay iseasily performed in a 96-well format, and hence is easily incorporatedinto a high-throughput screening assay. Cells are treated with testcompounds for the appropriate time course, lysed, the mRNA isreverse-transcribed, and the PCR is performed according to commonly usedmethods, (such as those described in Ausubel et al., Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., 1997), usingoligonucleotide primers that specifically hybridize with methioninesynthase nucleic acid. Changes in product levels of samples exposed totest compounds, relative to control samples, indicate test compoundsthat modulate methionine synthase expression.

Secondary Screens of Test Compounds that Appear to Modulate MethionineSynthase Activity

[0080] After test compounds that appear to have methioninesynthase-modulating activity are identified, it may be necessary ordesirable to subject these compounds to further testing. At late stagestesting will be performed in vivo to confirm that the compoundsinitially identified to affect methionine synthase activity will havethe predicted effect in vivo.

Test Compounds

[0081] In general, novel drugs for prevention of neural tube defects, orprevention and/or treatment of colon cancer or cardiovascular diseaseare identified from large libraries of both natural product or synthetic(or semi-synthetic) extracts or chemical libraries according to methodsknown in the art. Those skilled in the field of drug discovery anddevelopment will understand that the precise source of test extracts orcompounds is not critical to the screening procedure(s) of theinvention. Accordingly, virtually any number of chemical extracts orcompounds can be screened using the exemplary methods described herein.Examples of such extracts or compounds include, but are not limited to,plant-, fungal-, prokaryotic- or animal-based extracts, fermentationbroths, and synthetic compounds, as well as modification of existingcompounds. Numerous methods are also available for generating random ordirected synthesis (e.g., semi-synthesis or total synthesis) of anynumber of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available from BrandonAssociates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are produced, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.Furthermore, if desired, any library or compound is readily modifiedusing standard chemical, physical, or biochemical methods.

[0082] In addition, those skilled in the art of drug discovery anddevelopment readily understand that methods for dereplication (e.g.,taxonomic dereplication, biological dereplication, and chemicaldereplication, or any combination thereof) or the elimination ofreplicates or repeats of materials already known for their therapeuticactivities for homocysteinemia, megaloblastic anemia withoutmethylmalonic aciduria, cardiovasular disease, colon cancer, and neuraltube defects should be employed whenever possible.

[0083] When a crude extract is found to modulate methionine synthasebiological activity, further fractionation of the positive lead extractis necessary to isolate chemical constituents responsible for theobserved effect. Thus, the goal of the extraction, fractionation, andpurification process is the careful characterization and identificationof a chemical entity within the crude extract that modulates methioninesynthase biological activity. The same assays described herein for thedetection of activities in mixtures of compounds can be used to purifythe active component and to test derivatives thereof. Methods offractionation and purification of such heterogenous extracts are knownin the art. If desired, compounds shown to be useful agents fortreatment are chemically modified according to methods known in the art.Compounds identified as being of therapeutic value may be subsequentlyanalyzed using mammalian models of homocysteinemia, megaloblastic anemiawithout methylmalonic aciduria, cardiovasular disease, colon cancer, andneural tube defects.

Therapy

[0084] Compounds identified using any of the methods disclosed herein,may be administered to patients or experimental animals with apharmaceutically-acceptable diluent, carrier, or excipient, in unitdosage form. Conventional pharmaceutical practice may be employed toprovide suitable formulations or compositions to administer suchcompositions to patients or experimental animals. Although intravenousadministration is preferred, any appropriate route of administration maybe employed, for example, parenteral, subcutaneous, intramuscular,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, ororal administration. Therapeutic formulations may be in the form ofliquid solutions or suspensions; for oral administration, formulationsmay be in the form of tablets or capsules; and for intranasalformulations, in the form of powders, nasal drops, or aerosols.

[0085] Methods well known in the art for making formulations are foundin, for example, “Remington's Pharmaceutical Sciences.” Formulations forparenteral administration may, for example, contain excipients, sterilewater, or saline, polyalkylene glycols such as polyethylene glycol, oilsof vegetable origin, or hydrogenated naphthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for antagonists or agonists of the invention includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

EXAMPLES

[0086] The following examples are to illustrate, not limit theinvention.

Example 1 Cloning Human Methionine Synthase cDNA

[0087] An initial survey of the NCBI databases yielded several sequencescorresponding to methionine synthase from different organisms.Comparison of these sequences generated four very conserved regionsidentified as Boxes 1-4 in FIG. 1 (SEQ ID Nos:3-25). Degenerateoligonucleotides (SEQ ID Nos:26-66) were synthesized corresponding tothese conserved sequences (Table 1). These were used as primers forRT-PCR with human and mouse mRNA. These experiments yielded PCR productswhich were subcloned, sequenced and aligned as shown in FIG. 2. Insubsequent experiments, oligonucleotide primers were specified from thenon-degenerate internal sequences of the subclones and additional PCRproducts encompassing the conserved boxes were obtained. In laterexperiments, additional sequences were obtained by inverse PCR (“PCR”,FIG. 2) to obtain upstream or downstream sequences from those alreadydetermined. At the 3′ end, a mouse sequence was obtained from the dbESTdatabase (Accession Number W33307). This sequence was used as the sourceof primers for additional PCR experiments. Throughout the experiments,the sequences of the PCR products were considered provisionallyauthentic if they were homologous to the methionine synthase sequencesobtained from the databases. The sequences were taken as error free bycomparison of the sequences of at least two, and usually three,independent PCR reactions. Sequences were linked into a common sequenceif RT-PCRs bridging independently isolated sequences were successful.Through this approach the complete coding sequence was determinedthrough exclusive use of PCR reactions.

[0088] The coding sequence of human methionine synthase contains 3795 bp(SEQ ID NO:1) encoding a polypeptide of 1265 amino acids in length (SEQID NO:2) (FIG. 3), exceeding the length of published methioninesynthases by 11-29 residues. The putative initiation codon is in asequence of good context for the initiation of translation in eukaryoticcells (GACAACATGT, underlined nucleotides matching Kozak consensus(Kozak,M. (1991) J. Biol. Chem., 266, 19867-19870)). The predicted MW ofmethionine synthase is 141,000, comparing favorably with the publishedsize of 151,000 based on SDS-polyacrylamide electrophoresis of the pigenzyme (Chen, Z., Crippen, K., Gulati, S., and Baneijee, R. (1994) J.Biol. Chem., 269, 27193-27197). It shares 58% identity with the E. coliand 65% identity with the C. elegans enzyme.

Example 2 Chromosomal Location

[0089] Using FISH, the gene encoding methionine synthase was mapped tochromosome band 1q43, close to the telomeric region of the long arm(FIG. 4). A total of 50 cells with at least one signal were observed. Asignal was seen on 1 chromatid in 26 cells, on two chromatids in 14cells, on 3 chromatids in 7 cells, and on 4 chromatids in 3 cells. Theseresults confirm the previous assignment of the gene to chromosome 1 byMellman et al. (Mellman, I. S., Lin, P. F., Ruddle, F. H., andRosenberg, L. E. (1979) Proc. Natl. Acad. Sci. USA, 76, 405-409), whoused cobalamin binding as a marker for the enzyme in human-hamsterhybrids.

Example 3 Mutations in the cblG Complementation Group

[0090] Patients with deficiency of methionine synthase activity havebeen grouped into the cblG complementation group in cell fusionexperiments (Watkins, D. and Rosenblatt, D. S. (1988) J. Clin. Invest.,81, 1690-1694). Fibroblast cultures from patients assigned to cblG wereexamined by RT-PCR based SSCP analysis. Three mutations were identifiedby sequencing PCR fragments showing band shifts by SSCP (FIG. 5). Ineach case, the change was confirmed by an independent diagnostic test ongenomic DNA or a separate preparation of cDNA from patient fibroblasts.One of the mutations, 2756A→G (D919G), was confirmed by a diagnostictest that monitored the presence of a HaeIII site created by themutation (FIG. 5A). Using this test, it was identified as a polymorphismsince it was seen in 8 of 52 control alleles (15%). In two other cases,candidate deleterious mutations were identified. One is a 3 bp deletion,bp 2640-2642, that results in the deletion of an isoleucine codon(ΔIle881). It was confirmed by heteroduplex analysis of cDNA generatedby RT-PCR (FIG. 5B). The second is a point mutation, 2758C→G. It resultsin the amino acid substitution H920D. It was confirmed in genomic DNA bythe loss of a Sau96I site (FIG. 5C). The latter two mutations wereheterozygous in the patient cell lines. Their second mutation has notbeen identified. The candidate deleterious mutations were not seen inpanels of 68 or 52 control alleles, respectively.

Example 4 Additional Roles for Methionine Synthase Polymorphism(Asp919Gly or D919G) in Disease

[0091] The following data suggest that the D919G polymorphismcontributes to altered metabolism of homocysteine, methionine, folates,Vit. B12, and S-adenosylmethionine.

[0092] First in a Montreal study (n=303), in which mother-child pairs(cases and controls) were examined, we observed that infants who werehomozygous for the polymorphism (Gly/Gly; Table 2) were at decreasedrisk for NTD.

[0093] Measurements of serum folate, RBC folate, plasma homocysteine andserum cobalamin did not give any statistically significant differences,except the trend was toward low folate levels in Gly/Gly individuals(cases and controls).

[0094] A second study (n=255) in California also examined the methioninesynthase polymorphism as a risk factor for neural tube defects (Table3). This study shows a similar decreased risk of neural tube defects inchildren homozygous for Gly/Gly. Since the study encompassed a mix ofwhites and Hispanics, the data were reexamined stratified according toethnic origin. Both groups showed a protective effect of Gly/Gly.

[0095] In summary, two independent studies suggest a protective effectof Gly/Gly against the risk of neural tube defects. This is likely to bemediated by a mild reduction in methionine synthase activity.

[0096] Next, in a study of colon cancer, (212 cases and 345 controls),we observed a decreased risk for colon cancer in the individuals whowere homozygous for the polymorphism (relative risk=0.62); see Table 4.In the same study, we observed significantly decreased levels of plasmafolate in individuals who were homozygous for the polymorphism; seeTable 5.

[0097] The Boston study described in Tables 4 and 5 is presented againin Table 6 with the data stratified according to alcohol intake. Asshown in the table, Gly/Gly individuals with a low to medium alcoholintake had a relative risk associated with colon cancer of 0.11. Thecombined data (low+high alcohol) gave a risk level of 0.62 (Table 6).

[0098] In summary, drug therapy targeted to a reduction in methioninesynthase activity may be protective in individuals at risk for coloncancer or at risk for neural tube defects. Additional polymorphisms ormutations may also exert a protective effect against the risk of neuraltube defects or colon cancer. Conversely, it is understood that somepolymorphisms and/or mutations may enhance the risk of neural tubedefects or colon cancer, for example, by increasing methionine synthaseactivity.

Example 5 Role of Polymorphism on Homocysteine and Folate Levels

[0099] Third, in a study of individuals participating in the U.S. NHLBIFamily Heart Study, we observed both an increase in plasma homocysteinefollowing a methionine load and a decrease in plasma folate inindividuals who were homozygous for this polymorphism; see Table 7.

Example 6 Methionine Synthase Assays for the Detection of Compounds thatModulate Methionine Synthase Activity and Expression

[0100] Potentially useful therapeutic compounds that modulate (e.g.increase or decrease) methionine synthase activity or expression may beisolated by various screens that are well-known to those skilled in theart. Such compounds may modulate methionine synthase expression at thepre- or post-transcriptional level, or at the pre- or post-translationallevel.

Example 7 Materials and Methods

[0101] Cell lines. The skin fibroblast lines are from patients withmethionine synthase deficiency. They were assigned to the cblGcomplementation group in cell fusion experiments assayed by¹⁴C-methyltetrahydrofolate incorporation into cellular macromolecules(Watkins, D. and Rosenblatt, D. S. (1988) J. Clin. Invest., 81,1690-1694). Control fibroblasts were from other laboratory stocks or theMontreal Children's Hospital Cell Repository for Mutant Human CellStrains. Of the patients for which non-polymorphic mutations were found,WG 1892, a Caucasian male, was diagnosed at the age of 4 years withdevelopmental delay, tremors, gait instability, megaloblastic anemia andhomocystinuria; and WG2290, also a Caucasian male, was diagnosed at age3 months with failure to thrive, severe eczema, megaloblastic anemia andsurprisingly both homocystinuria and methylmalonic aciduria.

[0102] Materials. The T/A cloning kit was from Invitrogen. The GenecleanIII Kit was obtained from Bio 101 Inc. and the Wizard Mini-Preps werefrom Promega. The random-primed DNA labelling kit was fromBoehringer-Mannheim. Taq polymerase, Superscript II reversetranscriptase, AMV reverse transcriptase, Trizol reagent, DNAzolreagent, T4 DNA ligase, and restriction enzymes were purchased fromGibco BRL. The Sequenase kit for manual sequencing was from UnitedStates Biochemicals. The α-[³⁵S]dATP (12.5 Ci/mole) was from Dupont orICN. The oligonucleotide primers were synthesized by R. Clarizio of theMontreal Children's Hospital Research Institute OligonucleotideSynthesis Facility or the Sheldon Biotechnology Centre, McGillUniversity.

[0103] Homology matches. Comparisons were made between the published E.coli cobalamin-dependent methionine synthase sequence and sequences inthe NCBI databases (dbEST and GenBank) using the BLAST programs.

[0104] PCR cloning and DNA sequencing. DNA was prepared from fibroblastpellets by the method of Hoar et al. (Hoar,D. I., Haslam,D. B., andStarozik,D. M. (1984) Prenat. Diag., 4, 241-247). Total cellular RNA wasisolated by the method of Chirgwin et al. (Chirgwin,J. M., Przybyla,A.E., MacDonald,R. J., and Rutter,W. J. (1979) Biochemistry, 18,5294-5299) and is reverse-transcribed using oligo-dT₁₅ as primer. PCRwas conducted using degenerate oligonucleotides as primers, paired so asto link the sequences of different homology boxes. The PCRs wereconducted as described previously (Triggs-Raine,B. L., Akerman,B. R.,Clarke,J. T., and Gravel,R. A. (1991) Am. J. Hum. Genet., 49, 1041-1054)except that the temperature of incubation was modified to accommodatethe use of reduced temperatures in the annealing step or by step-downPCR (Hecker,K. H. and Roux,K. H. (1996) Biotechniques, 20, 478-485.(Abstract)). In some experiments, inverse PCR was used to determinesequence upstream or downstream of known sequence (Ochman,H., Medhora,M.M., Garza,D., and Hartl,D. L. (1990) PCR Protocols: A Guide to Methodsand Applications, Academic Press, San Diego, pp. 219-227). In theseinstances, genomic DNA or cDNA prepared by reverse transcription of RNAwas digested with different four base restriction endonucleases, ligatedwith T4 DNA ligase, and amplified by PCR using adjacent oligonucleotidespriming in opposite directions. Templates for inverse PCR at the cDNAlevel were generated with 12.5 μg RNA reversed transcribed using AMV-RT.Second strand synthesis was carried out using the random-primed DNAlabelling kit adding 1 μl of each dNTP. Samples were incubated 30 min.at 37° C. Template was then treated as genomic DNA for digestion andligation. Inverse PCR was used to obtain the 5′ and 3′ ends of the cDNAand to define an intron sequence adjacent to a splice junction for thedesign of a mutation diagnostic test. The PCR products were purifiedwith Geneclean and were subcloned in the pCR2.1 vector and transformedinto E. coli as per the supplier's protocol (TA Cloning Kit). Thecandidate clones were sequenced manually or by the DNA Core Facility ofthe Canadian Genetic Diseases Network or the McGill University SheldonBiotechnology Centre.

[0105] Mutation analysis. Genomic DNA and RNA were isolated from controlor patient fibroblast pellets using the DNAzol or Trizol reagents,respectively, as per the manufacturer. The cDNA template for PCR wasprepared by reverse transcription of 3-5 μg total RNA in reactionscontaining 400 U of Superscript II reverse transcriptase and 100 ngrandom hexamers in a total reaction volume of 20 ul. SSCP analysis wasperformed as described previously (Triggs-Raine,B. L., Akerman,B. R.,Clarke,J. T., and Gravel,R. A. (1991) Am. J. Hum. Genet., 49, 1041-1054)in reactions containing 4 μl of template, 1 μl of each dTTP, dCTP, dGTP(0.625 mM), 0.5 μl of dATP (0.625 mM), 1 μl α-[³⁵S]-dATP (12.5 Ci/mole).The radio labelled PCR products mixed with sequencing stop solution wereheat denatured and quick chilled on ice prior to loading(Triggs-Raine,B. L., Akerman,B. R., Clarke,J. T., and Gravel,R. A.(1991) Am. J. Hum. Genet., 49, 1041-1054). As well, an aliquot of eachsample was run without prior heating to identify the duplex product. Thefragments were subjected to electrophoresis in a 6% acrylamide/10%glycerol gel in 1×TBE for 18 hrs at 8 watts at room temperature. The gelwas dried and exposed to Biomax film (Kodak). Fragments that displayedband shifts were sequenced directly.

[0106] Two mutations were confirmed directly in PCR amplificationproducts from genomic DNA and one mutation was confirmed in reversedtranscribed mRNA. The PCR reactions for mutation confirmation wereperformed using 4 μl of cDNA template or 500 ng genomic DNA, 500 ng ofspecific primers, 2.5 U Taq polymerase and 1.5 mM MgCl2 in a 50 μlvolume. Heteroduplex analysis was accomplished by preheating PCRproducts to 95° C. for five minutes and subjecting the samples toelectrophoresis in a 9% polyacrylamide gel (Triggs-Raine,B. L.,Akerman,B. R., Clarke,J. T., and Gravel,R. A. (1991) Am. J. Hum. Genet.,49, 1041-1054). Other diagnostic assays were accomplished by digesting a15 μl sample of the PCR products with the indicated restrictionendonuclease prior to electrophoresis.

[0107] Chromosomal localization. Human metaphase spreads were obtainedfrom short-term cultures of phytohemaglutinin-stimulated peripheralblood lymphocytes. The cells were synchronized with thymidine andtreated with BrdU during the late S-phase before harvesting forsimultaneous observation of the hybridized sites and chromosome banding.The protocol for FISH was essentially as described previously (Lemieux,N., Malfoy, B., and Forrest, G. L. (1993) Genomics, 15, 169-172; Zhang,X. X., Rozen, R., Hediger, M. A., Goodyer, P., and Eydoux, P. (1994)Genomics, 24, 413-414). Briefly, a 5 kb DNA fragment of the methioninesynthase genomic DNA (generated by PCR using oligonucleotides #1782 and#1780) was labelled by nick translation with biotin-16-dUTP(Boehringer-Mannheim), ethanol precipitated and dissolved inhybridization buffer at a final concentration of 8 ng/μl. The slideswere denatured in 70% formamide, 2×SSC at 70° C. for 2 min. Thebiofinylated probe was denatured in the hybridization buffer at 95° C.for 10 min, quickly cooled on ice, then applied on slides. Post-washingwas done by rinsing in 50% formamide, 2×SSC at 37° C. The slides wereincubated with rabbit antibiotin antibody (Enzo Biochemicals),biotinylated goat anti-rabbit antibodies (BRL) and streptavidin-FITC.They were stained with propidium iodide and mounted inp-phenylenediamine, pH 11. Cells were observed under the microscope(Zeiss), then captured through a CCD camera and processed using a FISHsoftware (Applied Imaging). TABLE 1 Oligonucleotides used for cDNAcloning, chromosome mapping and mutation detection. Oligonucleotides^(a)Sequence Location^(b) D1729 5′-GAYGGNGCNATGGGNACNATGATHCA (SEQ ID NO:26)100-125 D1730 5′-GCNACNGTNAARGGNGAYGTNCAYGAYAT (SEQ ID NO:27) 2332-2360D1731 5′-RTTYTTNCCDATRTCRTGNACRTCNCCYTT (SEQ ID NO:28) 2370-2341 D17335′-RTGNAGRTAYTCNGCRAANGCYTCNGC (SEQ ID NO:29) 3426-3400 D17545′-ATRTGRTCNGGNGTNGTNCCRCARCANCCNCC (SEQ ID NO:30) 992-961 D17555′-GGNGGNTGYTGYGGNACNACNCCNGAYCAYAT (SEQ ID NO:31) 961-992 M1806A5′-GTCTGTGTCATAGCCCAGAATGGG (SEQ ID NO:32) 3795-3772 M1806B5′-TCAGTCTGTGTCATAGCCCAGAAT (SEQ ID NO:33) 3798-3775 305A5′-GAACTAGAAGACAGAAATTCTCTA (SEQ ID NO:34) (intronic) 407A5′-TTCCGAGGTCAGGAATTTAAAGATCA (SEQ ID NO:35) 151-176 407B5′-GTGTTCTTCGTTTAGCTTCTCCCG (SEQ ID NO:36) 150-127 407D5′-CCCCAGCCAGCAAGTATTCCTTAT (SEQ ID NO:37) 268-245 1107A5′-CTAGGTTGTATTTCCTTGAGGATC (SEQ ID NO:38) 3856-3833 1406D5′-GGAGCTGGAAAAATGTTTCTACCTC (SEQ ID NO:39) 2170-2194 1406E5′-ACAGGAGGGAAGAAAGTCATTCAG (SEQ ID NO:40) 1963-1986 1706A5′-CCTTCAATTATATTGAGAGGTCGGG (SEQ ID NO:41) 2129-2105 1707A5′-CAACCCGAAGGTCTGAAGAAAACC (SEQ ID NO:42) 28-51 1707B5′-CCCGCGCTCCAAGACCTGTCG (SEQ ID NO:43)  7-27 1707C5′-CGACAGGTCTTGGAGCGCGGG (SEQ ID NO:44) 27-7  17585′-GGAGTCATGACTCCTAAATCAATAACTC (SEQ ID NO:45) 2432-2405 17605′-GACGACTACAGCAGCATCATGGT (SEQ ID NO:46) 3355-3377 17665′-AAAAATCATTTCATCCAGGGAA (SEQ ID NO:47) 2526-2505 17725′-ATAGGCAAGAACATAGTTGGAGTAGT (SEQ ID NO:48) 2359-2384 17735′-TTTCATCTAACAGCTGGGAACACAC (SEQ ID NO:49) 2698-2674 17745′-TGCCTCTCAGACTTCATCGCTCCC (SEQ ID NO:50) 3241-3264 17805′-TGCAGCCTGGGGCACAGCAGC (SEQ ID NO:51) 3168-3148 17825′-ATGGATTGGCTGTCTGAACCTCAC (SEQ ID NO:52) 2824-2847 17965′-CATGGAAGAATATGAAGATATTAGAC (SEQ ID NO:53) 2727-2752 18035′-ACCATCATCCTCATAGGCCTTGCT (SEQ ID NO:54) 3354-3331 1806C5′-CAGACCTGCGAAGGTTGCGGTAC (SEQ ID NO:55) 3482-3504 1806F5′-GAAGTGGTTGCTCCTCCAATCAAC (SEQ ID NO:56) 2591-2568 18085′-GAGCAGCTTTCAGTATCTTATCACAT (SEQ ID NO:57) 2458-2433 18275′-ACAAGTTGTGTTCCTCCATTCCAGT (SEQ ID NO:58) 1657-1633 18285′-AGAGCGCTGTAATGTTGCAGGATCA (SEQ ID NO:59) 1125-1149 1907B5′-TGTTTTTCAATGCCCTTCACAAGGG (SEQ ID NO:60) 2057-2033 1907C5′-TAAAAAGTATGGAGCTGCTATGGTG (SEQ ID NO:61) 1464-1488 2606A5′-GACCAGACAGTAACATATGTCCTTC (SEQ ID NO:62) 1078-1054 2606B5′-ACATTACAGCGCTCTCCAATGTTAAC (SEQ ID NO:63) 1139-1114 2706A5′-TGAGGTTGAGAAATGGCTTGGACC (SEQ ID NO:64) 3750-3773 2706B5′-GCCACAGATATGTTCTTCCTCAATG (SEQ ID NO:65) 3749-3725 3107A5′-TGTGGAGAGCACGTCTTCTCTGCC (SEQ ID NO:66) −55-−32

[0108] TABLE 2 MS Polymorphism in Neural Tube Defects - Montreal StudyCase Con- Control Cases mothers trols mothers Odds Genotype N % N % N %N % ratio* 95% C.I. Asp/Asp 38 69 40 66 59 61 55 61 Asp/Gly 17 31 20 3328 29 34 38 Gly/Gly 0 0.9 1 2 10 10 1 1 0.07 0.004-1.29 N 55 61 97 90

[0109] TABLE 3 MS Polymorphisms in Neural Tube Defects - CaliforniaStudy Genotype Cases Controls Odds Ethnic Group 2756A-G N % N % ratio*95% C.I. Overall Asp/Asp 64 67 104 64 1.0 Asp/Gly 30 32 49 30 0.990.56-1.72 Gly/Gly 1 1 7 4 0.23 0.05-1.92 White only Asp/Asp 21 66 38 662.0 Asp/Gly 10 31 16 28 1.1 0.44-2.9  Gly/Gly 1 3 3 5 0.60 0.11-5.6 Hispanic only Asp/Asp 43 68 66 63 1.0 Asp/Gly 20 32 33 31 0.9 0.45-1.8 Gly/Gly 0 0 4 4 0

[0110] TABLE 4 Frequency of MS genotype and relative risk (RR) ofcolorectal cancer by MS genotype Cases Controls MS Genotype n % n % RR95% CI Asp/Asp 145 (68) 234 (68) 1.0  Asp/Gly  61 (29)  95 (28) 1.020.69-1.50 Gly/Gly  6  (3)  16  (5) 0.62 0.24-1.64 Total 212 345

[0111] TABLE 5 Mean of homocysteine and folate (geometric) by casecontrol status and MS Genotype in a colon cancer study Cases & CasesControls Controls MS genotype n mean n mean n mean Folate (Bio-Kit)ng/ml Asp/Asp 115  3.8 201  3.9 * 316  3.9 ** Asp/Gly 49  4.1 * 80 3.8 * 129  3.9 ** Gly/Gly 6  2.1 12  2.3 18  2.2 Homocysteine (μM)Asp/Asp 66 12.5 160 12.1 226 12.3 Asp/Gly 30 10.8 50 11.6 80 11.2Gly/Gly 4 13.4 9 11.7 13 12.5

[0112] TABLE 6 Age Adjusted Relative Risk of Colon Cancer According toMS Polymorphism and Alcohol Intake Status Among US Physicians Genotype2756A−>G Cases Controls Odds Alcohol intake Asp919Gly N N ratio 95% C.I.Low-Medium Asp/Asp 1013 2e+09 1.0 0-0.8 drinks/day Asp/Gly 7113 0.870.54-1.4  Gly/Gly 9 0.11 0.01-0.82 N High Asp/Asp 3721 7e+06 0.740.46-1.19  1-2+ drinks/day Asp/Gly 563 1.15 0.60-2.18 Gly/Gly 3.83 0.72-20.47 N

[0113] TABLE 7 Mean Homocysteine and Folate Status by MS Genotype (Dateof Analysis: Feb. 18, 1997) Methionine Synthase Genotype Asp/Asp Asp/GlyGly/Gly Result Result P Result P N 252 111 17 Fasting Hcy (μM) 8.5 8.40.76 8.7 post-methionine load 17.9 19.6 0.74 Hcy (μM) 7.4 0.05* 6.9 22.3Folate (microb test) 0.37 0.03* 6.3 0.37

[0114]

1 76 1 3919 DNA Homo sapiens Other (1)...(3919) Entire cloned cDNAencoding wild type methionine synthase. 1 ggtcacctgt ggagagcacgtcttctctgc cgcgccctct gcgcaaggag gagactcgac 60 aacatgtcac ccgcgctccaagacctgtcg caacccgaag gtctgaagaa aaccctgcgg 120 gatgagatca atgccattctgcagaagagg attatggtgc tggatggagg gatggggacc 180 atgatccagc gggagaagctaaacgaagaa cacttccgag gtcaggaatt taaagatcat 240 gccaggccgc tgaaaggcaacaatgacatt ttaagtataa ctcagcctga tgtcatttac 300 caaatccata aggaatacttgctggctggg gcagatatca ttgaaacaaa tacttttagc 360 agcactagta ttgcccaagctgactatggc cttgaacact tggcctaccg gatgaacatg 420 tgctctgcag gagtggccagaaaagctgcc gaggaggtaa ctctccagac aggaattaag 480 aggtttgtgg caggggctctgggtccgact aataagacac tctctgtgtc cccatctgtg 540 gaaaggccgg attataggaacatcacattt gatgagcttg ttgaagcata ccaagagcag 600 gccaaaggac ttctggatggcggggttgat atcttactca ttgaaactat ttttgatact 660 gccaatgcca aggcagccttgtttgcactc caaaatcttt ttgaggagaa atatgctccc 720 cggcctatct ttatttcagggacgatcgtt gataaaagtg ggcggactct ttccggacag 780 acaggagagg gatttgtcatcagcgtgtct catggagaac cactctgcat tggattaaat 840 tgtgctttgg gtgcagctgagatgagacct tttattgaaa taattggaaa atgtacaaca 900 gcctatgtcc tctgttatcccaatgcaggt cttcccaaca cctttggtga ctatgatgaa 960 acgccttcta tgatggccaagcacctaaag gattttgcta tggatggctt ggtcaatata 1020 gttggaggat gctgtgggtcaacaccagat catatcaggg aaattgctga agctgtgaaa 1080 aattgtaagc ctagagttccacctgccact gcttttgaag gacatatgtt actgtctggt 1140 ctagagccct tcaggattggaccgtacacc aactttgtta acattggaga gcgctgtaat 1200 gttgcaggat caaggaagtttgctaaactc atcatggcag gaaactatga agaagccttg 1260 tgtgttgcca aagtgcaggtggaaatggga gcccaggtgt tggatgtcaa catggatgat 1320 ggcatgctag atggtccaagtgcaatgacc agattttgca acttaattgc ttccgagcca 1380 gacatcgcaa aggtacctttgtgcatcgac tcctccaatt ttgctgtgat tgaagctggg 1440 ttaaagtgct gccaagggaagtgcattgtc aatagcatta gtctgaagga aggagaggac 1500 gacttcttgg agaaggccaggaagattaaa aagtatggag ctgctatggt ggtcatggct 1560 tttgatgaag aaggacaggcaacagaaaca gacacaaaaa tcagagtgtg cacccgggcc 1620 taccatctgc ttgtgaaaaaactgggcttt aatccaaatg acattatttt tgaccctaat 1680 atcctaacca ttgggactggaatggaggaa cacaacttgt atgccattaa ttttatccat 1740 gcaacaaaag tcattaaagaaacattacct ggagccagaa taagtggagg tctttccaac 1800 ttgtccttct ccttccgaggaatggaagcc attcgagaag caatgcatgg ggttttcctt 1860 taccatgcaa tcaagtctggcatggacatg gagatagtga atgctggaaa cctccctgtg 1920 tatgatgata tccataaggaacttctgcag ctctgtgaag atctcatctg gaataaagac 1980 cctgaggcca ctgagaagctcttacgttat gcccagactc aaggcacagg agggaagaaa 2040 gtcattcaga ctgatgagtggagaaatggc cctgtcgaag aacgccttga gtatgccctt 2100 gtgaagggca ttgaaaaacatattattgag gatactgagg aagccaggtt aaaccaaaaa 2160 aaatatcccc gacctctcaatataattgaa ggacccctga tgaatggaat gaaaattgtt 2220 ggtgatcttt ttggagctggaaaaatgttt ctacctcagg ttataaagtc agcccgggtt 2280 atgaagaagg ctgttggccaccttatccct ttcatggaaa aagaaagaga agaaaccaga 2340 gtgcttaacg gcacagtagaagaagaggac ccttaccagg gcaccatcgt gctggccact 2400 gttaaaggcg acgtgcacgacataggcaag aacatagttg gagtagtcct tggctgcaat 2460 aatttccgag ttattgatttaggagtcatg actccatgtg ataagatact gaaagctgct 2520 cttgaccaca aagcagatataattggcctg tcaggactca tcactccttc cctggatgaa 2580 atgatttttg ttgccaaggaaatggagaga ttagctataa ggattccatt gttgattgga 2640 ggagcaacca cttcaaaaacccacacagca gttaaaatag ctccgagata cagtgcacct 2700 gtaatccatg tcctggacgcgtccaagagt gtggtggtgt gttcccagct gttagatgaa 2760 aatctaaagg atgaatactttgaggaaatc atggaagaat atgaagatat tagacaggac 2820 cattatgagt ctctcaaggagaggagatac ttacccttaa gtcaagccag aaaaagtggt 2880 ttccaaatgg attggctgtctgaacctcac ccagtgaagc ccacgtttat tgggacccag 2940 gtctttgaag actatgacctgcagaagctg gtggactaca ttgactggaa gcctttcttt 3000 gatgtctggc agctccggggcaagtacccg aatcgaggct tccccaagat atttaacgac 3060 aaaacagtag gtggagaggccaggaaggtc tacgatgatg cccacaatat gctgaacaca 3120 ctgattagtc aaaagaaactccgggcccgg ggtgtggttg ggttctggcc agcacagagt 3180 atccaagacg acattcacctgtacgcagag gctgctgtgc cccaggctgc agagcccata 3240 gccactttct atgggttaaggcaacaggct gagaaggact ctgccagcac ggagccatac 3300 tactgcctct cagacttcatcgctcccttg cattctggca tccgtgacta cctgggcctg 3360 tttgccgttg cctgctttggggtagaagag ctgagcaagg cctatgagga tgatggtgac 3420 gactacagca gcatcatggtcaaggcgctg ggggaccggc tggcagaggc ctttgcagaa 3480 gagctccatg aaagagttcgccgagaactg tgggcctact gtggcagtga gcagctggac 3540 gtcgcagacc tgcgaaggttgcggtacaag ggcatccgcc cggctcctgg ctaccccagc 3600 cagcccgacc acaccgagaagctcaccatg tggagactcg cagacatcga gcagtctaca 3660 ggcattaggt taacagaatcattagcaatg gcacctgctt cagcagtctc aggcctctac 3720 ttctccaatt tgaagtccaaatattttgct gtggggaaga tttccaagga tcaggttgag 3780 gattatgcat tgaggaagaacatatctgtg gctgaggttg agaaatggct tggacccatt 3840 ttgggatatg atacagactaactttttttt ttttttttgc cttttttatc ttgatgatcc 3900 tcaaggaaat acaacctag3919 2 1265 PRT Homo sapiens VARIANT (1)...(1265) Wild type methioninesynthase polypeptide. 2 Met Ser Pro Ala Leu Gln Asp Leu Ser Gln Pro GluGly Leu Lys Lys 1 5 10 15 Thr Leu Arg Asp Glu Ile Asn Ala Ile Leu GlnLys Arg Ile Met Val 20 25 30 Leu Asp Gly Gly Met Gly Thr Met Ile Gln ArgGlu Lys Leu Asn Glu 35 40 45 Glu His Phe Arg Gly Gln Glu Phe Lys Asp HisAla Arg Pro Leu Lys 50 55 60 Gly Asn Asn Asp Ile Leu Ser Ile Thr Gln ProAsp Val Ile Tyr Gln 65 70 75 80 Ile His Lys Glu Tyr Leu Leu Ala Gly AlaAsp Ile Ile Glu Thr Asn 85 90 95 Thr Phe Ser Ser Thr Ser Ile Ala Gln AlaAsp Tyr Gly Leu Glu His 100 105 110 Leu Ala Tyr Arg Met Asn Met Cys SerAla Gly Val Ala Arg Lys Ala 115 120 125 Ala Glu Glu Val Thr Leu Gln ThrGly Ile Lys Arg Phe Val Ala Gly 130 135 140 Ala Leu Gly Pro Thr Asn LysThr Leu Ser Val Ser Pro Ser Val Glu 145 150 155 160 Arg Pro Asp Tyr ArgAsn Ile Thr Phe Asp Glu Leu Val Glu Ala Tyr 165 170 175 Gln Glu Gln AlaLys Gly Leu Leu Asp Gly Gly Val Asp Ile Leu Leu 180 185 190 Ile Glu ThrIle Phe Asp Thr Ala Asn Ala Lys Ala Ala Leu Phe Ala 195 200 205 Leu GlnAsn Leu Phe Glu Glu Lys Tyr Ala Pro Arg Pro Ile Phe Ile 210 215 220 SerGly Thr Ile Val Asp Lys Ser Gly Arg Thr Leu Ser Gly Gln Thr 225 230 235240 Gly Glu Gly Phe Val Ile Ser Val Ser His Gly Glu Pro Leu Cys Ile 245250 255 Gly Leu Asn Cys Ala Leu Gly Ala Ala Glu Met Arg Pro Phe Ile Glu260 265 270 Ile Ile Gly Lys Cys Thr Thr Ala Tyr Val Leu Cys Tyr Pro AsnAla 275 280 285 Gly Leu Pro Asn Thr Phe Gly Asp Tyr Asp Glu Thr Pro SerMet Met 290 295 300 Ala Lys His Leu Lys Asp Phe Ala Met Asp Gly Leu ValAsn Ile Val 305 310 315 320 Gly Gly Cys Cys Gly Ser Thr Pro Asp His IleArg Glu Ile Ala Glu 325 330 335 Ala Val Lys Asn Cys Lys Pro Arg Val ProPro Ala Thr Ala Phe Glu 340 345 350 Gly His Met Leu Leu Ser Gly Leu GluPro Phe Arg Ile Gly Pro Tyr 355 360 365 Thr Asn Phe Val Asn Ile Gly GluArg Cys Asn Val Ala Gly Ser Arg 370 375 380 Lys Phe Ala Lys Leu Ile MetAla Gly Asn Tyr Glu Glu Ala Leu Cys 385 390 395 400 Val Ala Lys Val GlnVal Glu Met Gly Ala Gln Val Leu Asp Val Asn 405 410 415 Met Asp Asp GlyMet Leu Asp Gly Pro Ser Ala Met Thr Arg Phe Cys 420 425 430 Asn Leu IleAla Ser Glu Pro Asp Ile Ala Lys Val Pro Leu Cys Ile 435 440 445 Asp SerSer Asn Phe Ala Val Ile Glu Ala Gly Leu Lys Cys Cys Gln 450 455 460 GlyLys Cys Ile Val Asn Ser Ile Ser Leu Lys Glu Gly Glu Asp Asp 465 470 475480 Phe Leu Glu Lys Ala Arg Lys Ile Lys Lys Tyr Gly Ala Ala Met Val 485490 495 Val Met Ala Phe Asp Glu Glu Gly Gln Ala Thr Glu Thr Asp Thr Lys500 505 510 Ile Arg Val Cys Thr Arg Ala Tyr His Leu Leu Val Lys Lys LeuGly 515 520 525 Phe Asn Pro Asn Asp Ile Ile Phe Asp Pro Asn Ile Leu ThrIle Gly 530 535 540 Thr Gly Met Glu Glu His Asn Leu Tyr Ala Ile Asn PheIle His Ala 545 550 555 560 Thr Lys Val Ile Lys Glu Thr Leu Pro Gly AlaArg Ile Ser Gly Gly 565 570 575 Leu Ser Asn Leu Ser Phe Ser Phe Arg GlyMet Glu Ala Ile Arg Glu 580 585 590 Ala Met His Gly Val Phe Leu Tyr HisAla Ile Lys Ser Gly Met Asp 595 600 605 Met Glu Ile Val Asn Ala Gly AsnLeu Pro Val Tyr Asp Asp Ile His 610 615 620 Lys Glu Leu Leu Gln Leu CysGlu Asp Leu Ile Trp Asn Lys Asp Pro 625 630 635 640 Glu Ala Thr Glu LysLeu Leu Arg Tyr Ala Gln Thr Gln Gly Thr Gly 645 650 655 Gly Lys Lys ValIle Gln Thr Asp Glu Trp Arg Asn Gly Pro Val Glu 660 665 670 Glu Arg LeuGlu Tyr Ala Leu Val Lys Gly Ile Glu Lys His Ile Ile 675 680 685 Glu AspThr Glu Glu Ala Arg Leu Asn Gln Lys Lys Tyr Pro Arg Pro 690 695 700 LeuAsn Ile Ile Glu Gly Pro Leu Met Asn Gly Met Lys Ile Val Gly 705 710 715720 Asp Leu Phe Gly Ala Gly Lys Met Phe Leu Pro Gln Val Ile Lys Ser 725730 735 Ala Arg Val Met Lys Lys Ala Val Gly His Leu Ile Pro Phe Met Glu740 745 750 Lys Glu Arg Glu Glu Thr Arg Val Leu Asn Gly Thr Val Glu GluGlu 755 760 765 Asp Pro Tyr Gln Gly Thr Ile Val Leu Ala Thr Val Lys GlyAsp Val 770 775 780 His Asp Ile Gly Lys Asn Ile Val Gly Val Val Leu GlyCys Asn Asn 785 790 795 800 Phe Arg Val Ile Asp Leu Gly Val Met Thr ProCys Asp Lys Ile Leu 805 810 815 Lys Ala Ala Leu Asp His Lys Ala Asp IleIle Gly Leu Ser Gly Leu 820 825 830 Ile Thr Pro Ser Leu Asp Glu Met IlePhe Val Ala Lys Glu Met Glu 835 840 845 Arg Leu Ala Ile Arg Ile Pro LeuLeu Ile Gly Gly Ala Thr Thr Ser 850 855 860 Lys Thr His Thr Ala Val LysIle Ala Pro Arg Tyr Ser Ala Pro Val 865 870 875 880 Ile His Val Leu AspAla Ser Lys Ser Val Val Val Cys Ser Gln Leu 885 890 895 Leu Asp Glu AsnLeu Lys Asp Glu Tyr Phe Glu Glu Ile Met Glu Glu 900 905 910 Tyr Glu AspIle Arg Gln Asp His Tyr Glu Ser Leu Lys Glu Arg Arg 915 920 925 Tyr LeuPro Leu Ser Gln Ala Arg Lys Ser Gly Phe Gln Met Asp Trp 930 935 940 LeuSer Glu Pro His Pro Val Lys Pro Thr Phe Ile Gly Thr Gln Val 945 950 955960 Phe Glu Asp Tyr Asp Leu Gln Lys Leu Val Asp Tyr Ile Asp Trp Lys 965970 975 Pro Phe Phe Asp Val Trp Gln Leu Arg Gly Lys Tyr Pro Asn Arg Gly980 985 990 Phe Pro Lys Ile Phe Asn Asp Lys Thr Val Gly Gly Glu Ala ArgLys 995 1000 1005 Val Tyr Asp Asp Ala His Asn Met Leu Asn Thr Leu IleSer Gln Lys 1010 1015 1020 Lys Leu Arg Ala Arg Gly Val Val Gly Phe TrpPro Ala Gln Ser Ile 1025 1030 1035 1040 Gln Asp Asp Ile His Leu Tyr AlaGlu Ala Ala Val Pro Gln Ala Ala 1045 1050 1055 Glu Pro Ile Ala Thr PheTyr Gly Leu Arg Gln Gln Ala Glu Lys Asp 1060 1065 1070 Ser Ala Ser ThrGlu Pro Tyr Tyr Cys Leu Ser Asp Phe Ile Ala Pro 1075 1080 1085 Leu HisSer Gly Ile Arg Asp Tyr Leu Gly Leu Phe Ala Val Ala Cys 1090 1095 1100Phe Gly Val Glu Glu Leu Ser Lys Ala Tyr Glu Asp Asp Gly Asp Asp 11051110 1115 1120 Tyr Ser Ser Ile Met Val Lys Ala Leu Gly Asp Arg Leu AlaGlu Ala 1125 1130 1135 Phe Ala Glu Glu Leu His Glu Arg Val Arg Arg GluLeu Trp Ala Tyr 1140 1145 1150 Cys Gly Ser Glu Gln Leu Asp Val Ala AspLeu Arg Arg Leu Arg Tyr 1155 1160 1165 Lys Gly Ile Arg Pro Ala Pro GlyTyr Pro Ser Gln Pro Asp His Thr 1170 1175 1180 Glu Lys Leu Thr Met TrpArg Leu Ala Asp Ile Glu Gln Ser Thr Gly 1185 1190 1195 1200 Ile Arg LeuThr Glu Ser Leu Ala Met Ala Pro Ala Ser Ala Val Ser 1205 1210 1215 GlyLeu Tyr Phe Ser Asn Leu Lys Ser Lys Tyr Phe Ala Val Gly Lys 1220 12251230 Ile Ser Lys Asp Gln Val Glu Asp Tyr Ala Leu Arg Lys Asn Ile Ser1235 1240 1245 Val Ala Glu Val Glu Lys Trp Leu Gly Pro Ile Leu Gly TyrAsp Thr 1250 1255 1260 Asp 1265 3 9 PRT Escherichia coli 3 Asp Gly GlyMet Gly Thr Met Ile Gln 1 5 4 9 PRT Cyanobacterium synechocystis 4 AspGly Ala Met Gly Thr Asn Leu Gln 1 5 5 9 PRT Mycobacterium leprae 5 AspGly Ala Met Gly Thr Gln Leu Gln 1 5 6 9 PRT Hemophilus influenzae 6 AspGly Ala Met Gly Thr Met Ile Gln 1 5 7 9 PRT Caenorrhabditis elegans 7Asp Gly Ala Met Gly Thr Met Ile Gln 1 5 8 9 PRT Homo sapiens 8 Asp GlyGly Met Gly Thr Met Ile Gln 1 5 9 13 PRT Escherichia coli 9 Ala Thr ValLys Gly Asp Val His Asp Ile Gly Lys Asn 1 5 10 10 13 PRT Cyanobacteriumsynechocystis 10 Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn 1 510 11 13 PRT Mycobacterium leprae 11 Ala Thr Val Lys Gly Asp Val His AspIle Gly Lys Asn 1 5 10 12 13 PRT Hemophilus influenzae 12 Ala Thr ValLys Gly Asp Val His Asp Ile Gly Lys Asn 1 5 10 13 13 PRT Caenorrhabditiselegans 13 Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn 1 5 10 1413 PRT Homo sapiens 14 Ala Thr Val Lys Gly Asp Val His Asp Ile Gly LysAsn 1 5 10 15 10 PRT Escherichia coli 15 Leu Ala Glu Ala Phe Ala Glu TyrLeu His 1 5 10 16 10 PRT Cyanobacterium synechocystis 16 Met Ala Glu AlaLeu Ala Glu Trp Thr His 1 5 10 17 10 PRT Mycobacterium leprae 17 Leu ThrGlu Ala Leu Ala Glu Tyr Trp His 1 5 10 18 10 PRT Hemophilus influenzae18 Leu Ala Glu Ala Met Ala Glu Tyr Leu His 1 5 10 19 10 PRTCaenorrhabditis elegans 19 Leu Ala Glu Ala Tyr Ala Glu Tyr Leu His 1 510 20 10 PRT Homo sapiens 20 Leu Ala Glu Ala Phe Ala Glu Glu Leu His 1 510 21 11 PRT Escherichia coli 21 Gly Gly Cys Cys Gly Thr Thr Pro Gln HisIle 1 5 10 22 11 PRT Cyanobacterium synechocystis 22 Gly Gly Cys Cys GlyThr Arg Pro Asp His Ile 1 5 10 23 11 PRT Mycobacterium leprae 23 Gly GlyCys Cys Gly Thr Thr Pro Asp His Ile 1 5 10 24 11 PRT Caenorrhabditiselegans 24 Gly Gly Cys Cys Gly Thr Thr Pro Asp His Ile 1 5 10 25 11 PRTHomo sapiens 25 Gly Gly Cys Cys Gly Ser Thr Pro Asp His Ile 1 5 10 26 26DNA Homo sapiens variation (1)...(26) n is a, t, g, or c; h is a, c, ort; d is a, g, or t; and r is a or g; 26 gayggngcna tgggnacnat gathca 2627 29 DNA Homo sapiens variation (1)...(29) n is a, t, g, or c; h is a,c, or t; d is a, g, or t; and r is a or g; 27 gcnacngtna arggngaygtncaygayat 29 28 30 DNA Homo sapiens variation (1)...(30) n is a, t, g,or c; h is a, c, or t; d is a, g, or t; and r is a or g; 28 rttyttnccdatrtcrtgna crtcnccytt 30 29 27 DNA Homo sapiens variation (1)...(27) nis a, t, g, or c; h is a, c, or t; d is a, g, or t; and r is a or g; 29rtgnagrtay tcngcraang cytcngc 27 30 32 DNA Homo sapiens variation(1)...(32) n is a, t, g, or c; h is a, c, or t; d is a, g, or t; and ris a or g; 30 atrtgrtcng gngtngtncc rcarcanccn cc 32 31 32 DNA Homosapiens variation (1)...(32) n is a, t, g, or c; h is a, c, or t; d isa, g, or t; and r is a or g; 31 ggnggntgyt gyggnacnac nccngaycay at 3232 24 DNA Mus musculus 32 gtctgtgtca tagcccagaa tggg 24 33 24 DNA Musmusculus 33 tcagtctgtg tcatagccca gaat 24 34 24 DNA Homo sapiens 34gaactagaag acagaaattc tcta 24 35 26 DNA Homo sapiens 35 ttccgaggtcaggaatttaa agatca 26 36 24 DNA Homo sapiens 36 gtgttcttcg tttagcttctcccg 24 37 24 DNA Homo sapiens 37 ccccagccag caagtattcc ttat 24 38 24DNA Homo sapiens 38 ctaggttgta tttccttgag gatc 24 39 25 DNA Homo sapiens39 ggagctggaa aaatgtttct acctc 25 40 24 DNA Homo sapiens 40 acaggagggaagaaagtcat tcag 24 41 25 DNA Homo sapiens 41 ccttcaatta tattgagagg tcggg25 42 24 DNA Homo sapiens 42 caacccgaag gtctgaagaa aacc 24 43 21 DNAHomo sapiens 43 cccgcgctcc aagacctgtc g 21 44 21 DNA Homo sapiens 44cgacaggtct tggagcgcgg g 21 45 28 DNA Homo sapiens 45 ggagtcatgactcctaaatc aataactc 28 46 23 DNA Homo sapiens 46 gacgactaca gcagcatcatggt 23 47 22 DNA Homo sapiens 47 aaaaatcatt tcatccaggg aa 22 48 26 DNAHomo sapiens 48 ataggcaaga acatagttgg agtagt 26 49 25 DNA Homo sapiens49 tttcatctaa cagctgggaa cacac 25 50 24 DNA Homo sapiens 50 tgcctctcagacttcatcgc tccc 24 51 21 DNA Homo sapiens 51 tgcagcctgg ggcacagcag c 2152 24 DNA Homo sapiens 52 atggattggc tgtctgaacc tcac 24 53 26 DNA Homosapiens 53 catggaagaa tatgaagata ttagac 26 54 24 DNA Homo sapiens 54accatcatcc tcataggcct tgct 24 55 23 DNA Homo sapiens 55 cagacctgcgaaggttgcgg tac 23 56 24 DNA Homo sapiens 56 gaagtggttg ctcctccaat caac24 57 26 DNA Homo sapiens 57 gagcagcttt cagtatctta tcacat 26 58 25 DNAHomo sapiens 58 acaagttgtg ttcctccatt ccagt 25 59 25 DNA Homo sapiens 59agagcgctgt aatgttgcag gatca 25 60 25 DNA Homo sapiens 60 tgtttttcaatgcccttcac aaggg 25 61 25 DNA Homo sapiens 61 taaaaagtat ggagctgctatggtg 25 62 25 DNA Homo sapiens 62 gaccagacag taacatatgt ccttc 25 63 26DNA Homo sapiens 63 acattacagc gctctccaat gttaac 26 64 24 DNA Homosapiens 64 tgaggttgag aaatggcttg gacc 24 65 25 DNA Homo sapiens 65gccacagata tgttcttcct caatg 25 66 24 DNA Homo sapiens 66 tgtggagagcacgtcttctc tgcc 24 67 50 PRT Homo sapiens 67 Leu Ala Thr Val Lys Gly AspVal His Asp Ile Gly Lys Asn Ile Val 1 5 10 15 Gly Val Val Leu Gly CysAsn Asn Phe Arg Val Ile Asp Leu Gly Val 20 25 30 Met Thr Pro Cys Asp LysIle Leu Lys Ala Ala Leu Asp His Lys Ala 35 40 45 Asp Ile 50 68 50 PRTMus musculus 68 Leu Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys AsnIle Val 1 5 10 15 Gly Val Val Leu Ala Cys Asn Asn Phe Arg Val Ile AspLeu Gly Val 20 25 30 Met Thr Pro Cys Asp Lys Ile Leu Gln Ala Ala Leu AspHis Lys Ala 35 40 45 Asp Ile 50 69 50 PRT Cyanobacterium synechocystis69 Ile Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Leu Val 1 510 15 Asp Ile Ile Leu Ser Asn Asn Gly Tyr Arg Val Val Asn Leu Gly Ile 2025 30 Lys Gln Pro Val Glu Asn Ile Ile Glu Ala Tyr Lys Lys His Arg Pro 3540 45 Asp Cys 50 70 50 PRT Mycobacterium leprae 70 Leu Ala Thr Val LysGly Asp Val His Asp Ile Gly Lys Asn Leu Val 1 5 10 15 Asp Ile Ile LeuSer Asn Asn Gly Tyr Glu Val Val Asn Leu Gly Ile 20 25 30 Lys Gln Pro IleThr Asn Ile Leu Glu Val Ala Glu Asp Lys Ser Ala 35 40 45 Asp Val 50 7150 PRT Caenorrhabditis elegans 71 Ile Ala Thr Val Lys Gly Asp Val HisAsp Ile Gly Lys Asn Ile Val 1 5 10 15 Ser Val Val Leu Gly Cys Asn AsnPhe Lys Val Val Asp Leu Gly Val 20 25 30 Met Thr Pro Cys Glu Asn Ile IleLys Ala Ala Ile Glu Glu Lys Ala 35 40 45 Asp Phe 50 72 50 PRT Hemophilusinfluenzae 72 Ile Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys AsnIle Val 1 5 10 15 Ser Val Val Met Gln Cys Asn Asn Phe Glu Val Ile AspLeu Gly Val 20 25 30 Met Val Pro Ala Asp Lys Ile Ile Gln Thr Ala Ile AsnGln Lys Thr 35 40 45 Asp Ile 50 73 50 PRT Escherichia coli 73 Ile AlaThr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Ile Val 1 5 10 15 GlyVal Val Leu Gln Cys Asn Asn Tyr Glu Ile Val Asp Leu Gly Val 20 25 30 MetVal Pro Ala Glu Lys Ile Leu Arg Thr Ala Lys Glu Val Asn Ala 35 40 45 AspLeu 50 74 1265 PRT Homo sapiens VARIANT (1)...(1265) Xaa at position 881is either Ile or no amino acid; Xaa at position 919 is either Asp orGly; Xaa at position 920 is either His or Asp. 74 Met Ser Pro Ala LeuGln Asp Leu Ser Gln Pro Glu Gly Leu Lys Lys 1 5 10 15 Thr Leu Arg AspGlu Ile Asn Ala Ile Leu Gln Lys Arg Ile Met Val 20 25 30 Leu Asp Gly GlyMet Gly Thr Met Ile Gln Arg Glu Lys Leu Asn Glu 35 40 45 Glu His Phe ArgGly Gln Glu Phe Lys Asp His Ala Arg Pro Leu Lys 50 55 60 Gly Asn Asn AspIle Leu Ser Ile Thr Gln Pro Asp Val Ile Tyr Gln 65 70 75 80 Ile His LysGlu Tyr Leu Leu Ala Gly Ala Asp Ile Ile Glu Thr Asn 85 90 95 Thr Phe SerSer Thr Ser Ile Ala Gln Ala Asp Tyr Gly Leu Glu His 100 105 110 Leu AlaTyr Arg Met Asn Met Cys Ser Ala Gly Val Ala Arg Lys Ala 115 120 125 AlaGlu Glu Val Thr Leu Gln Thr Gly Ile Lys Arg Phe Val Ala Gly 130 135 140Ala Leu Gly Pro Thr Asn Lys Thr Leu Ser Val Ser Pro Ser Val Glu 145 150155 160 Arg Pro Asp Tyr Arg Asn Ile Thr Phe Asp Glu Leu Val Glu Ala Tyr165 170 175 Gln Glu Gln Ala Lys Gly Leu Leu Asp Gly Gly Val Asp Ile LeuLeu 180 185 190 Ile Glu Thr Ile Phe Asp Thr Ala Asn Ala Lys Ala Ala LeuPhe Ala 195 200 205 Leu Gln Asn Leu Phe Glu Glu Lys Tyr Ala Pro Arg ProIle Phe Ile 210 215 220 Ser Gly Thr Ile Val Asp Lys Ser Gly Arg Thr LeuSer Gly Gln Thr 225 230 235 240 Gly Glu Gly Phe Val Ile Ser Val Ser HisGly Glu Pro Leu Cys Ile 245 250 255 Gly Leu Asn Cys Ala Leu Gly Ala AlaGlu Met Arg Pro Phe Ile Glu 260 265 270 Ile Ile Gly Lys Cys Thr Thr AlaTyr Val Leu Cys Tyr Pro Asn Ala 275 280 285 Gly Leu Pro Asn Thr Phe GlyAsp Tyr Asp Glu Thr Pro Ser Met Met 290 295 300 Ala Lys His Leu Lys AspPhe Ala Met Asp Gly Leu Val Asn Ile Val 305 310 315 320 Gly Gly Cys CysGly Ser Thr Pro Asp His Ile Arg Glu Ile Ala Glu 325 330 335 Ala Val LysAsn Cys Lys Pro Arg Val Pro Pro Ala Thr Ala Phe Glu 340 345 350 Gly HisMet Leu Leu Ser Gly Leu Glu Pro Phe Arg Ile Gly Pro Tyr 355 360 365 ThrAsn Phe Val Asn Ile Gly Glu Arg Cys Asn Val Ala Gly Ser Arg 370 375 380Lys Phe Ala Lys Leu Ile Met Ala Gly Asn Tyr Glu Glu Ala Leu Cys 385 390395 400 Val Ala Lys Val Gln Val Glu Met Gly Ala Gln Val Leu Asp Val Asn405 410 415 Met Asp Asp Gly Met Leu Asp Gly Pro Ser Ala Met Thr Arg PheCys 420 425 430 Asn Leu Ile Ala Ser Glu Pro Asp Ile Ala Lys Val Pro LeuCys Ile 435 440 445 Asp Ser Ser Asn Phe Ala Val Ile Glu Ala Gly Leu LysCys Cys Gln 450 455 460 Gly Lys Cys Ile Val Asn Ser Ile Ser Leu Lys GluGly Glu Asp Asp 465 470 475 480 Phe Leu Glu Lys Ala Arg Lys Ile Lys LysTyr Gly Ala Ala Met Val 485 490 495 Val Met Ala Phe Asp Glu Glu Gly GlnAla Thr Glu Thr Asp Thr Lys 500 505 510 Ile Arg Val Cys Thr Arg Ala TyrHis Leu Leu Val Lys Lys Leu Gly 515 520 525 Phe Asn Pro Asn Asp Ile IlePhe Asp Pro Asn Ile Leu Thr Ile Gly 530 535 540 Thr Gly Met Glu Glu HisAsn Leu Tyr Ala Ile Asn Phe Ile His Ala 545 550 555 560 Thr Lys Val IleLys Glu Thr Leu Pro Gly Ala Arg Ile Ser Gly Gly 565 570 575 Leu Ser AsnLeu Ser Phe Ser Phe Arg Gly Met Glu Ala Ile Arg Glu 580 585 590 Ala MetHis Gly Val Phe Leu Tyr His Ala Ile Lys Ser Gly Met Asp 595 600 605 MetGlu Ile Val Asn Ala Gly Asn Leu Pro Val Tyr Asp Asp Ile His 610 615 620Lys Glu Leu Leu Gln Leu Cys Glu Asp Leu Ile Trp Asn Lys Asp Pro 625 630635 640 Glu Ala Thr Glu Lys Leu Leu Arg Tyr Ala Gln Thr Gln Gly Thr Gly645 650 655 Gly Lys Lys Val Ile Gln Thr Asp Glu Trp Arg Asn Gly Pro ValGlu 660 665 670 Glu Arg Leu Glu Tyr Ala Leu Val Lys Gly Ile Glu Lys HisIle Ile 675 680 685 Glu Asp Thr Glu Glu Ala Arg Leu Asn Gln Lys Lys TyrPro Arg Pro 690 695 700 Leu Asn Ile Ile Glu Gly Pro Leu Met Asn Gly MetLys Ile Val Gly 705 710 715 720 Asp Leu Phe Gly Ala Gly Lys Met Phe LeuPro Gln Val Ile Lys Ser 725 730 735 Ala Arg Val Met Lys Lys Ala Val GlyHis Leu Ile Pro Phe Met Glu 740 745 750 Lys Glu Arg Glu Glu Thr Arg ValLeu Asn Gly Thr Val Glu Glu Glu 755 760 765 Asp Pro Tyr Gln Gly Thr IleVal Leu Ala Thr Val Lys Gly Asp Val 770 775 780 His Asp Ile Gly Lys AsnIle Val Gly Val Val Leu Gly Cys Asn Asn 785 790 795 800 Phe Arg Val IleAsp Leu Gly Val Met Thr Pro Cys Asp Lys Ile Leu 805 810 815 Lys Ala AlaLeu Asp His Lys Ala Asp Ile Ile Gly Leu Ser Gly Leu 820 825 830 Ile ThrPro Ser Leu Asp Glu Met Ile Phe Val Ala Lys Glu Met Glu 835 840 845 ArgLeu Ala Ile Arg Ile Pro Leu Leu Ile Gly Gly Ala Thr Thr Ser 850 855 860Lys Thr His Thr Ala Val Lys Ile Ala Pro Arg Tyr Ser Ala Pro Val 865 870875 880 Xaa His Val Leu Asp Ala Ser Lys Ser Val Val Val Cys Ser Gln Leu885 890 895 Leu Asp Glu Asn Leu Lys Asp Glu Tyr Phe Glu Glu Ile Met GluGlu 900 905 910 Tyr Glu Asp Ile Arg Gln Xaa Xaa Tyr Glu Ser Leu Lys GluArg Arg 915 920 925 Tyr Leu Pro Leu Ser Gln Ala Arg Lys Ser Gly Phe GlnMet Asp Trp 930 935 940 Leu Ser Glu Pro His Pro Val Lys Pro Thr Phe IleGly Thr Gln Val 945 950 955 960 Phe Glu Asp Tyr Asp Leu Gln Lys Leu ValAsp Tyr Ile Asp Trp Lys 965 970 975 Pro Phe Phe Asp Val Trp Gln Leu ArgGly Lys Tyr Pro Asn Arg Gly 980 985 990 Phe Pro Lys Ile Phe Asn Asp LysThr Val Gly Gly Glu Ala Arg Lys 995 1000 1005 Val Tyr Asp Asp Ala HisAsn Met Leu Asn Thr Leu Ile Ser Gln Lys 1010 1015 1020 Lys Leu Arg AlaArg Gly Val Val Gly Phe Trp Pro Ala Gln Ser Ile 1025 1030 1035 1040 GlnAsp Asp Ile His Leu Tyr Ala Glu Ala Ala Val Pro Gln Ala Ala 1045 10501055 Glu Pro Ile Ala Thr Phe Tyr Gly Leu Arg Gln Gln Ala Glu Lys Asp1060 1065 1070 Ser Ala Ser Thr Glu Pro Tyr Tyr Cys Leu Ser Asp Phe IleAla Pro 1075 1080 1085 Leu His Ser Gly Ile Arg Asp Tyr Leu Gly Leu PheAla Val Ala Cys 1090 1095 1100 Phe Gly Val Glu Glu Leu Ser Lys Ala TyrGlu Asp Asp Gly Asp Asp 1105 1110 1115 1120 Tyr Ser Ser Ile Met Val LysAla Leu Gly Asp Arg Leu Ala Glu Ala 1125 1130 1135 Phe Ala Glu Glu LeuHis Glu Arg Val Arg Arg Glu Leu Trp Ala Tyr 1140 1145 1150 Cys Gly SerGlu Gln Leu Asp Val Ala Asp Leu Arg Arg Leu Arg Tyr 1155 1160 1165 LysGly Ile Arg Pro Ala Pro Gly Tyr Pro Ser Gln Pro Asp His Thr 1170 11751180 Glu Lys Leu Thr Met Trp Arg Leu Ala Asp Ile Glu Gln Ser Thr Gly1185 1190 1195 1200 Ile Arg Leu Thr Glu Ser Leu Ala Met Ala Pro Ala SerAla Val Ser 1205 1210 1215 Gly Leu Tyr Phe Ser Asn Leu Lys Ser Lys TyrPhe Ala Val Gly Lys 1220 1225 1230 Ile Ser Lys Asp Gln Val Glu Asp TyrAla Leu Arg Lys Asn Ile Ser 1235 1240 1245 Val Ala Glu Val Glu Lys TrpLeu Gly Pro Ile Leu Gly Tyr Asp Thr 1250 1255 1260 Asp 1265 75 3856 DNAHomo sapiens variation (1)...(3856) nnn at positions 2640-2642 is eitherAAT or no nucleotides; n at position 2756 is either A or G; n atposition 2758 is either C or G. 75 atgtcacccg cgctccaaga cctgtcgcaacccgaaggtc tgaagaaaac cctgcgggat 60 gagatcaatg ccattctgca gaagaggattatggtgctgg atggagggat ggggaccatg 120 atccagcggg agaagctaaa cgaagaacacttccgaggtc aggaatttaa agatcatgcc 180 aggccgctga aaggcaacaa tgacattttaagtataactc agcctgatgt catttaccaa 240 atccataagg aatacttgct ggctggggcagatatcattg aaacaaatac ttttagcagc 300 actagtattg cccaagctga ctatggccttgaacacttgg cctaccggat gaacatgtgc 360 tctgcaggag tggccagaaa agctgccgaggaggtaactc tccagacagg aattaagagg 420 tttgtggcag gggctctggg tccgactaataagacactct ctgtgtcccc atctgtggaa 480 aggccggatt ataggaacat cacatttgatgagcttgttg aagcatacca agagcaggcc 540 aaaggacttc tggatggcgg ggttgatatcttactcattg aaactatttt tgatactgcc 600 aatgccaagg cagccttgtt tgcactccaaaatctttttg aggagaaata tgctccccgg 660 cctatcttta tttcagggac gatcgttgataaaagtgggc ggactctttc cggacagaca 720 ggagagggat ttgtcatcag cgtgtctcatggagaaccac tctgcattgg attaaattgt 780 gctttgggtg cagctgagat gagaccttttattgaaataa ttggaaaatg tacaacagcc 840 tatgtcctct gttatcccaa tgcaggtcttcccaacacct ttggtgacta tgatgaaacg 900 ccttctatga tggccaagca cctaaaggattttgctatgg atggcttggt caatatagtt 960 ggaggatgct gtgggtcaac accagatcatatcagggaaa ttgctgaagc tgtgaaaaat 1020 tgtaagccta gagttccacc tgccactgcttttgaaggac atatgttact gtctggtcta 1080 gagcccttca ggattggacc gtacaccaactttgttaaca ttggagagcg ctgtaatgtt 1140 gcaggatcaa ggaagtttgc taaactcatcatggcaggaa actatgaaga agccttgtgt 1200 gttgccaaag tgcaggtgga aatgggagcccaggtgttgg atgtcaacat ggatgatggc 1260 atgctagatg gtccaagtgc aatgaccagattttgcaact taattgcttc cgagccagac 1320 atcgcaaagg tacctttgtg catcgactcctccaattttg ctgtgattga agctgggtta 1380 aagtgctgcc aagggaagtg cattgtcaatagcattagtc tgaaggaagg agaggacgac 1440 ttcttggaga aggccaggaa gattaaaaagtatggagctg ctatggtggt catggctttt 1500 gatgaagaag gacaggcaac agaaacagacacaaaaatca gagtgtgcac ccgggcctac 1560 catctgcttg tgaaaaaact gggctttaatccaaatgaca ttatttttga ccctaatatc 1620 ctaaccattg ggactggaat ggaggaacacaacttgtatg ccattaattt tatccatgca 1680 acaaaagtca ttaaagaaac attacctggagccagaataa gtggaggtct ttccaacttg 1740 tccttctcct tccgaggaat ggaagccattcgagaagcaa tgcatggggt tttcctttac 1800 catgcaatca agtctggcat ggacatggagatagtgaatg ctggaaacct ccctgtgtat 1860 gatgatatcc ataaggaact tctgcagctctgtgaagatc tcatctggaa taaagaccct 1920 gaggccactg agaagctctt acgttatgcccagactcaag gcacaggagg gaagaaagtc 1980 attcagactg atgagtggag aaatggccctgtcgaagaac gccttgagta tgcccttgtg 2040 aagggcattg aaaaacatat tattgaggatactgaggaag ccaggttaaa ccaaaaaaaa 2100 tatccccgac ctctcaatat aattgaaggacccctgatga atggaatgaa aattgttggt 2160 gatctttttg gagctggaaa aatgtttctacctcaggtta taaagtcagc ccgggttatg 2220 aagaaggctg ttggccacct tatccctttcatggaaaaag aaagagaaga aaccagagtg 2280 cttaacggca cagtagaaga agaggacccttaccagggca ccatcgtgct ggccactgtt 2340 aaaggcgacg tgcacgacat aggcaagaacatagttggag tagtccttgg ctgcaataat 2400 ttccgagtta ttgatttagg agtcatgactccatgtgata agatactgaa agctgctctt 2460 gaccacaaag cagatataat tggcctgtcaggactcatca ctccttccct ggatgaaatg 2520 atttttgttg ccaaggaaat ggagagattagctataagga ttccattgtt gattggagga 2580 gcaaccactt caaaaaccca cacagcagttaaaatagctc cgagatacag tgcacctgtn 2640 nnccatgtcc tggacgcgtc caagagtgtggtggtgtgtt cccagctgtt agatgaaaat 2700 ctaaaggatg aatactttga ggaaatcatggaagaatatg aagatattag acaggncnat 2760 tatgagtctc tcaaggagag gagatacttacccttaagtc aagccagaaa aagtggtttc 2820 caaatggatt ggctgtctga acctcacccagtgaagccca cgtttattgg gacccaggtc 2880 tttgaagact atgacctgca gaagctggtggactacattg actggaagcc tttctttgat 2940 gtctggcagc tccggggcaa gtacccgaatcgaggcttcc ccaagatatt taacgacaaa 3000 acagtaggtg gagaggccag gaaggtctacgatgatgccc acaatatgct gaacacactg 3060 attagtcaaa agaaactccg ggcccggggtgtggttgggt tctggccagc acagagtatc 3120 caagacgaca ttcacctgta cgcagaggctgctgtgcccc aggctgcaga gcccatagcc 3180 actttctatg ggttaaggca acaggctgagaaggactctg ccagcacgga gccatactac 3240 tgcctctcag acttcatcgc tcccttgcattctggcatcc gtgactacct gggcctgttt 3300 gccgttgcct gctttggggt agaagagctgagcaaggcct atgaggatga tggtgacgac 3360 tacagcagca tcatggtcaa ggcgctgggggaccggctgg cagaggcctt tgcagaagag 3420 ctccatgaaa gagttcgccg agaactgtgggcctactgtg gcagtgagca gctggacgtc 3480 gcagacctgc gaaggttgcg gtacaagggcatccgcccgg ctcctggcta ccccagccag 3540 cccgaccaca ccgagaagct caccatgtggagactcgcag acatcgagca gtctacaggc 3600 attaggttaa cagaatcatt agcaatggcacctgcttcag cagtctcagg cctctacttc 3660 tccaatttga agtccaaata ttttgctgtggggaagattt ccaaggatca ggttgaggat 3720 tatgcattga ggaagaacat atctgtggctgaggttgaga aatggcttgg acccattttg 3780 ggatatgata cagactaact tttttttttttttttgcctt ttttatcttg atgatcctca 3840 aggaaataca acctag 3856 76 10 DNAHomo sapiens 76 gacaacatgt 10

What is claimed is:
 1. A substantially pure human nucleic acidcomprising at least 40 nucleotides that hybridizes under high stringencyconditions to a sequence found within the nucleic acid of SEQ ID NO:1.2. The nucleic acid of claim 1, wherein said sequence has a sequencecomplementary to at least 50% of at least 60 contiguous nucleotides ofthe nucleic acid encoding the methionine synthase polypeptide, saidsequence sufficient to allow nucleic acid hybridization under highstringency conditions.
 3. The nucleic acid of claim 1, wherein saidnucleic acid comprises a mutation or a polymorphism, wherein saidnucleic acid probe detects a mutation or polymorphism selected from thegroup consisting of D919G, H920D, and ΔIle881.
 4. The nucleic acid ofclaim 3, wherein said sequence of said nucleic acid comprises thecobalamin binding domain of the human methionine synthase gene.
 5. Thenucleic acid of claim 2, wherein at least 18 contiguous nucleotides ofsaid sequence are complementary to at least 90% of the correspondingnucleotides of the nucleic acid encoding the methionine synthasepolypeptide.
 6. The nucleic acid of claim 1, wherein said highstringency conditions comprise hybridization in 2×SSC at 40° C.
 7. Asubstantially pure human nucleic acid, wherein the sequence of saidnucleic acid is at least 75% identical to the corresponding region of atleast 50 contiguous base pairs of the nucleic acid of SEQ ID NO:1.
 8. Asubstantially pure human nucleic acid, wherein the sequence of saidnucleic acid is at least 35% identical to the corresponding region of atleast 50 contiguous base pairs of the nucleic acid of SEQ ID NO:1.
 9. Akit for the analysis of a human methionine synthase nucleic acid, saidkit comprising a nucleic acid probe useful for detecting in the nucleicacids of a human a mutation or polymorphism in said methionine synthasenucleic acid, wherein said mutation or polymorphism is selected from thegroup consisting of D919G, H920D, and ΔIle881.
 10. The kit of claim 9,wherein said probe comprises at least 40 nucleotides that hybridizes athigh stringency to a sequence found within the nucleic acid of SEQ IDNO:1.